Androgen-regulated PMEPA1 gene and polypeptides

ABSTRACT

This invention relates to the androgen-regulated gene, PMEPA1, and proteins encoded by this gene, including variants and analogs thereof. Also provided are other androgen-regulated nucleic acids, a polynucleotide array containing these androgen-regulated nucleic acids, and methods of using the polynucleotide array in the diagnosis and prognosis of prostate cancer.

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] The present application is a continuation-in-part of copendingU.S. application Ser. No. 10/390,045, filed Mar. 18, 2003, which is adivisional of U.S. Applicaton Ser. No. 09/769,482, filed Jan. 26, 2001,allowed, which is based upon U.S. provisional applications S. No.60/178,772, and 60/179,045, filed Jan. 28, 2000, and Jan. 31, 2000,respectively, priority to which is claimed under 35 U.S.C. § 119(e). Theentire disclosures of these applications are expressly incorporatedherein by reference.

GOVERNMENT INTEREST

[0002] The invention described herein may be manufactured, licensed, andused for governmental purposes without payment of royalties to usthereon.

FIELD OF THE INVENTION

[0003] The present invention relates to tumor suppressor genes, and inparticular, PMEPA1 genes, and the proteins encoded by these genes,including variants and/or analogs thereof. More particularly, thepresent invention is based in part on the discovery that PMEPA1polypeptides inhibit cancer cell growth. The present invention alsorelates to novel, androgen-regulated nucleic acids, polynucleotidearrays containing androgen-regulated nucleic acids, such as PMEPA1, andmethods of using the array in the evaluation of hormone-related cancers,such as prostate cancer.

BACKGROUND

[0004] Prostate cancer (CaP) is the most common malignancy in Americanmen and second leading cause of cancer mortality (1). Serum-prostatespecific antigen (PSA) tests have revolutionized the early detection ofCaP (2). Although PSA has revolutionized early detection of prostatecancer, there is still a very high false positive rate. The increasingincidence of CaP has translated into wider use of radical prostatectomyas well as other therapies for localized disease (3-5). The widespectrum of biologic behavior (6) exhibited by prostatic neoplasms posesa difficult problem in predicting the clinical course for the individualpatient (3-5). Traditional prognostic markers such as grade, clinicalstage, and pretreatment PSA have limited prognostic value for individualmen (3-5). A more reliable technique for the evaluation and prognosis ofCaP is desirable.

[0005] Molecular studies have shown a significant heterogeneity betweenmultiple cancer foci present in a cancerous prostate gland (7, 8). Thesestudies have also documented that the metastatic lesion can arise fromcancer foci other than those present in dominant tumors (7).Approximately 50-60% of patients treated with radical prostatectomy forlocalized prostate carcinomas are found to have microscopic disease thatis not organ-confined, and a significant portion of these patientsrelapse (9). Therefore, identification and characterization of geneticalterations defining CaP onset and progression is crucial inunderstanding the biology and clinical course of the disease.

[0006] Despite recent intensive research investigations, much remains tobe learned about specific molecular defects associated with CaP onsetand progression (6, 10-15). Alterations of the tumor suppressor genep53, bcl-2 and the androgen receptor (AR), are frequently reported inadvanced CaP (6, 10-15). However, the exact role of these geneticdefects in the genesis and progression of CaP is poorly understood (6,10-15). Recent studies have shown that the “focal p53 immunostaining” orbcl-2 immunostaining in radical prostatectomy specimens were independentprognostic markers for cancer recurrence after surgery (16-19).Furthermore, the combination of p53 and bcl-2 alterations was a strongerpredictor of cancer recurrence after radical prostatectomy (18).

[0007] The roles of several new chromosome loci harboring putativeproto-oncogenes or tumor suppressor genes are being currently evaluatedin CaP (7-13). High frequency of allelic losses on 8p21-22, 7q31.1,10q23-25 and 16q24 loci have been shown in CaP (6, 10-15). PTEN1/MMAC1,a recently discovered tumor suppressor gene on chromosome 10q25, isfrequently altered in advanced CaP (20, 21). Gains of chromosome 8q24harboring c-myc and prostate stem-cell antigen (PSCA) genes have alsobeen shown in prostate cancer (22, 23). Studies utilizing comparativegenomic hybridization (CGH) have shown frequent losses of novelchromosomal loci including 2q, 5q and 6q and gains of 11p, 12q, 3q, 4qand 2p in CaP (24, 25). The inventors have recently mapped a 1.5megabase interval at 6q16-21 which may contain the putative tumorsuppressor gene involved in a subset of prostate tumors. The risk for 6qLOH to non-organ confined disease was five fold higher than for organconfined disease (26). Chromosome regions, 1q24-25 and Xq27-28 have beenlinked to familial CaP (27, 28).

[0008] It is evident that multiple molecular approaches need to beexplored to identify CaP-associated genetic alterations. Emergingstrategies for defining cancer specific genetic alterations andcharacterizing androgen regulated genes in rat prostate and LNCaP humanprostate cancer cell models include, among others, the study of globalgene expression profiles in cancer cells and corresponding normal cellsby differential display (DD) (29) and more recent techniques, such asserial amplification of gene expression (SAGE) (30) and DNA micro-arrays(31; U.S. Pat. Nos. 5,744,305 and 5,837,832 which are hereinincorporated by reference) followed by targeted analyses of promisingcandidates. Our laboratory has also employed DD, SAGE and DNAmicroarrays to study CaP associated gene expression alterations (32-33).Each of these techniques, however, is limited. The number of transcriptsthat can be analyzed is the major limitation encountered in subtractivehybridization and differential display approaches. Furthermore, whilecDNA microarray approaches can determine expression of a large number ofgenes in a high throughput manner, the current limitations of cDNAarrays include the presence of specific arrays used for analyses and theinability to discover novel genes.

[0009] While alterations of critical tumor-suppressor genes andoncogenes are important in prostate tumorogenesis, it is also recognizedthat hormonal mechanisms play equally important roles in prostatetumorogenesis. The cornerstone of therapy in patients with metastaticdisease is androgen ablation, commonly referred to as “hormonal therapy(34),” which is dependent on the inhibition of androgen signaling inprostate cancer cells. Androgen ablation can be achieved, for example,by orchiectomy, by the administration of estrogen, or more recently byone of the luteinizing hormone-releasing hormone agonists. Recentclinical trials have demonstrated the efficacy of combining anantiandrogen to orchiectomy or a luteinizing hormone-releasing hormoneto block the remaining androgens produced by the adrenal glands.Although approximately 80% of patients initially respond to hormonalablation, the vast majority of patients eventually relapse (35),presumably due to neoplastic clones of cells which become refractory tothis therapy.

[0010] Alterations of the androgen receptor gene by mutations in thehormone binding domain of the AR or by amplification of the AR gene havebeen reported in advanced stages of CaP. Much remains to be learned,however, about the molecular mechanisms of the AR-mediated cellsignaling in prostate growth and tumorogenesis (36-43). Our earlierstudies have also described mutations of the AR in a subset of CaP (40).Mutations of the AR are reported to modify the ligand (androgen) bindingof the AR by making the receptor promiscuous, so that it may bind toestrogen, progesterone, and related molecules, in addition to theandrogens (36, 38, 42). Altered ligand binding specificity of the mutantAR may provide one of the mechanisms for increased function in cancercells. Amplifications of the AR gene in hormone-refractory CaP representyet another scenario where increase in AR function is associated withtumor progression (44, 45).

[0011] Several growth factors commonly involved in cell proliferationand tumorogenesis, e.g., IGF1, EGF, and others, have been shown toactivate the transcription transactivation functions of the AR (46). Theco-activator of the AR transcription factor functions may also play arole in prostate cancer (47). Recent studies analyzing expression of theandrogen-regulated genes (ARGs) in hormone sensitive and refractoryCWR22 nude mice xenograft models (48) have also shown expression ofseveral androgen regulated genes in AR positive recurrent tumorsfollowing castration, suggesting activation of AR in these tumors (49).

[0012] In addition to the alterations of the androgen signalingpathway(s) in prostate tumor progression, androgen mechanisms aresuspected to play a role in the predisposition to CaP. Prolongedadministration of high levels of testosterone has been shown to induceCaP in rats (50-52). Although recent evidence suggests an association ofandrogen levels and risk of CaP, this specific observation remains to beestablished. (53). An independent line of investigations addressing thelength of inherited polyglutamine (CAG) repeat sequence in the AR geneand CaP risk have shown that men with shorter repeats were at high riskof distant metastasis and fatal CaP (54, 55). Moreover, the sizedistribution of AR CAG repeats in various ethnic groups has alsosuggested a possible relationship of shorter CAG repeats and increasedprostate cancer risks in African-American men (56, 57). Biochemicalexperiments evaluating AR-CAG repeat length and in vitro transcriptiontransactivation functions of the AR revealed that AR with shorter CAGrepeats possessed a more potent transcription transactivation activity(58). Thus, molecular epidemiologic studies and biochemicalexperimentation suggest that gain of AR function, consequently resultingin transcriptional transactivation of downstream targets of the AR gene,may play an important role in CaP initiation. However, downstreamtargets of AR must be defined in order to understand the biologic basisof these observations.

[0013] The biologic effects of androgen on target cells, e.g., prostaticepithelial cell proliferation and differentiation as well as theandrogen ablation-induced cell death, are likely mediated bytranscriptional regulation of ARGs by the androgen receptor (reviewed in59). Abrogation of androgen signaling resulting from structural changesin the androgen gene or functional alterations of AR due to modulationof AR functions by other proteins would have profound effects ontranscriptional regulation of genes regulated by AR and, thus, on thegrowth and development of the prostate gland, including abnormal growthcharacterized by benign prostatic hyperplasia and prostatic cancer. Thenature of ARGs in the context of CaP initiation and progression,however, remains largely unknown. Since forced proliferation of the ARprostate cancer cells lacking AR induces cell-death related phenotypes(60), the studies utilizing AR expression via heterologous promoters incell cultures have failed to address the observations relating to gainof AR functions and prostate cancer progression. Moreover, suitableanimal models to assess gain of AR functions do not exist. Therefore,the expression profile of androgen responsive genes (ARGs) has potentialto serve as read-out of the AR signaling status. Such a read-out mayalso define potential biomarkers for onset and progression of thoseprostate cancers which may involve abrogation of the androgen signalingpathway. Furthermore, functional analysis of androgen regulated geneswill help understand the biochemical components of the androgensignaling pathways.

SUMMARY OF THE INVENTION

[0014] The present invention relates to the identification andcharacterization of a novel androgen-regulated gene that exhibitsabundant expression in prostate tissue. The novel gene has beendesignated PMEPA1. Our work with PMEPA1 is further described in U.S.Provisional Application S. No. 60/378,949, filed May 10, 2002, and PCTApplication No. PCT/US03/XXXXX, filed May 9, 2003, the entiredisclosures of which are hereby incorporated by reference.

[0015] The invention provides the isolated nucleotide sequence of PMEPA1or fragments thereof and nucleic acid sequences that hybridize toPMEPA1. These sequences have utility, for example, as markers ofprostate cancer and other prostate-related diseases, and as targets fortherapeutic intervention in prostate cancer and other prostate-relateddiseases. The invention further provides a vector that directs theexpression of PMEPA1, and a host cell transfected or transduced withthis vector.

[0016] In another embodiment, the invention provides a method ofdetecting prostate cancer cells in a biological sample, for example, byusing nucleic acid amplification techniques with primers and probesselected to bind specifically to the PMEPA1 sequence.

[0017] In another aspect, the invention relates to an isolatedpolypeptide encoded by the PMEPA1 gene or a fragment thereof, andantibodies generated against the PMEPA1 polypeptide, peptides, orportions thereof, which can be used to detect, treat, and preventprostate cancer.

[0018] In another aspect, the invention provides variants of the PMEPA1polypeptide that retain at least one of the following abilities:inhibiting cancer cell growth, reducing the expression of an androgenreceptor, or modulating the expression of a gene whose transcription isregulated by the androgen receptor. In one embodiment, these variantsare at least 95% identical to SEQ ID NO:3 and inhibit the growth ofprostate cancer cells (e.g., LNCaP cells), as measured, for example, ina colony-forming assay.

[0019] In another aspect, the invention provides a method of inhibitingthe growth of a cancer cell, comprising administering these variants tothe cancer cell in an amount effective to inhibit the growth of thecancer cell. In one embodiment the cancer cell is a prostate cancercell. The polypeptide may be administered directly to the cell orindirectly using a vector containing a polynucleotide sequence thatencodes the variant. These methods include therapeutic methods oftreating cancer, and in particular, prostate cancer.

[0020] A further embodiment of the invention provides a method ofreducing the expression of an androgen receptor or modulating theexpression of genes that are transcriptionally regulated by androgenreceptor, including, but not limited to the prostate-specific antigen(PSA) gene, the PSMA gene, and the PCGEM1 gene. Thus, in one aspect, theinvention provides a method of reducing the expression in a cancer cellof an androgen receptor or modulating (i.e., increasing or decreasing)the expression of a gene whose transcription is regulated by theandrogen receptor, comprising administering the variants described aboveto the cancer cell, in an amount effective to reduce the androgenreceptor or modulate the expression of the gene in the cancer cell. Inone embodiment the cancer cell is a prostate cancer cell. Thepolypeptide may be administered directly to the cell or indirectly usinga vector containing a polynucleotide sequence that encodes the variant.

[0021] In yet another aspect, the invention provides variants of thePMEPA1 polypeptide having at least one mutation and/or deletion in theat least one of the PY motifs of PMEPA1, as discussed in further detailbelow. Such mutations reduce the cell growth inhibitory effects ofPMEPA1. These PMEPA1 variants can be used, for example, to definecellular proteins through which PMEPA1 interacts, directly orindirectly, to mediate cell growth inhibitory functions.

[0022] In a still further embodiment, the invention provides thepolynucleotides that encode the PMEPA1 variants, as well as methods (asdescribed above for a polypeptide comprising SEQ ID NO:3) of using thesevariants, for example, to inhibit cancer cell growth, including prostatecancer, and/or to reduce the expression of an androgen receptor and/orto modulate the expression of a gene whose transcription is regulated bythe androgen receptor.

[0023] The present invention also relates to a polynucleotide arraycomprising (a) a planar, non-porous solid support having at least afirst surface; and (b) a first set of polynucleotide probes attached tothe first surface of the solid support, where the first set ofpolynucleotide probes comprises polynucleotide sequences derived fromgenes that are up-regulated, such as PMEPA1, or down-regulated inresponse to androgen, including genes downstream of the androgenreceptor gene and genes upstream of the androgen receptor gene thatmodulate androgen receptor function. In another embodiment of theinvention the polynucleotides immobilized on the solid support includegenes that are known to be involved in testosterone biosynthesis andmetabolism. In another embodiment of the invention the oligonucleotidesimmobilized on the solid support include genes whose expression isaltered in prostate cancer or is specific to prostate tissue.

[0024] In another embodiment, the invention provides a method for thediagnosis or prognosis of prostate cancer, comprising (a) hybridizingnucleic acids of a target cell of a patient with a polynucleotide array,as described above, to obtain a first hybridization pattern, where thefirst hybridization pattern represents an expression profile ofandrogen-regulated genes in the target cell; (b) comparing the firsthybridization pattern of the target cell to a second hybridizationpattern, where the second hybridization pattern represents an expressionprofile of androgen-regulated genes in prostate cancer, and (c)diagnosing or prognosing prostate cancer in the patient.

[0025] Thus, a first aspect of the present invention is directed towardsa method for analysis of radical prostatectomy specimens for theexpression profile of those genes involved in androgen receptor-mediatedsignaling. In a preferred embodiment, computer models may be developedfor the analysis of expression profiles. Another aspect of the inventionis directed towards a method of correlating expression profiles withclinico-pathologic features. In a preferred embodiment, computer modelsto identify gene expression features associated with tumor phenotypesmay be developed. Another aspect of the invention is directed towards amethod of distinguishing indolent prostate cancers from those with amore aggressive phenotype. In a preferred embodiment, computer models tosuch cancers may be developed. Another aspect of the invention isdirected towards a method of analyzing tumor specimens of patientstreated by radical prostate surgery to help define prognosis. Anotheraspect of the invention is directed towards a method of screeningcandidate genes for the development of a blood test for improvedprostate cancer detection. Another aspect of the invention is directedtowards a method of identifying androgen regulated genes that may serveas biomarkers for response to treatment to screen drugs for thetreatment of advanced prostate cancer.

[0026] This invention is further directed to a method of identifying anexpression profile of androgen-regulated genes in a target cell,comprising hybridizing the nucleic acids of the target cell with apolynucleotide array, as described above, to obtain a hybridizationpattern, where the hybridization pattern represents the expressionprofile of androgen-regulated genes in the target cell.

[0027] Additional features and advantages of the invention will be setforth in the description which follows, and in part will be apparentfrom the description, or may be learned by practice of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

[0028]FIG. 1 is a Northern blot showing that PMEPA1 is expressed at highlevels in prostate tissue. Multiple tissue northern blots werehybridized with PMEPA1 and GAPDH probes. The arrows indicate the twovariants of the PMEPA1 transcript.

[0029]FIG. 2 shows the androgen-dependent expression of PMEPA1. FIG. 2Ais a Northern blot using PMEPA1 probe with mRNA derived from LNCaP cellswith or without R1881 treatment for various durations. FIG. 2B is aNorthern blot of PMEPA1 expression in primary epithelial cell culturesof normal prostate and prostate and breast cancer cell lines.

[0030] FIGS. 3A-H show the effect of PMEPA1 on colony formation.Prostate tumor cell lines: C4 (FIG. 3A), C4-2 (FIG. 3B), C4-2B (FIG.3C), LNCaP (FIG. 3D), DU145 (FIG. 3E), and PC3 (FIG. 3F) weretransfected with 3 μg of each of PMEPA1-V5-pcDNA3.1 (PMEPA1) andpcDNA3.1 vector (Vector) in triplicate sets. In a separate experimentsLNCaP (FIG. 3G) and PC3 (FIG. 3H) cells were transfected with controlvector or expression vectors encoding wt-PMEPA1 or PMEPA1-PY mutants (1.PMEPA1-V5-pcDNA3.1, 2. PMEPA1-PY1m-pcDNA3.1, 3. PMEPA1-PY2m-pcDNA3.1, 4.PMEPA1-PY1m/PY2 m-pcDNA3.1, and 5. pcDNA3.1). Transfected cells wereselected for plasmid-containing cells with G418 for 3 weeks andsurviving cells were fixed and stained with crystal violet. In eachexperiment, the number of colonies per dish were counted and displayedas histograms, representing the mean number of colonies±SD of thetriplicate sets. For each cell line, a photograph of one dish of cellstreated with 3 μg of each plasmid is also shown.

[0031]FIG. 4A shows PMEPA1-mediated down regulation of androgen receptorand its functional consequences on androgen receptor regulated genes.LNCaP cells stably transfected with PMEPA1-GFP and pEGFP (control)plasmids were cultured in medium with cFBS for 5 days and then werestimulated with R1881 at 0.1 nM. Cells were harvested for Westernblotting at 0 h, 12 h and 24 h after androgen stimulation. Antibodiesagainst androgen receptor, PSA, PSMA and tubulin were used to detectcorresponding proteins on Western Blots.

[0032]FIG. 4B shows that PMEPA1 does not reduce androgen receptorexpression through a non-specific, PMEPA1-induced effect on theubiquitin-proteasome pathway. Stable PMEPA1-GFP-Tet-LNCaP transfectants(Tet-off system) were cultured in proper medium with or withouttetracycline for 10 days and were applied for immunoblotting. Antibodiesagainst androgen receptor, GFP, p27 and tubulin were used to detect thecorresponding proteins.

[0033]FIG. 5 shows the effect of PMEPA1 on cell proliferation. StablePMEPA1-GFP-Tet-LNCaP transfectants were seeded in 96-well plates with orwithout 1 μg/ml of tetracycline in the medium. The cell proliferationwas measured using the CellTiter 96 Aqueous One Solution kit at theindicated time. Tet+ and Tet− denote the cell culture medium with orwithout tetracycline, respectively. The OD values reflecting the cellnumbers are significantly different (p<0.01) between the two groupsexcept on day one.

[0034]FIG. 6 defines binding of PMEPA1 to NEDD4 proteins. The in vitrotranscription/translation products ([³⁵S]Methionine-labeled lysates)derived from expression plasmids: PMEPA1-V5-pcDNA3.1 (Lanes 1, 5),PMEPA1-PY1m-pcDNA3.1 (Lanes 2, 6), PMEPA1-PY2m-pcDNA3.1 (Lanes 3, 7),and PMEPA1-PY1m/PY2m-pcDNA3.1 (Lanes 4, 8) were incubated withGST-NEDD4-WW-Sepharose beads (Lanes 1-4) or control GST beads (Lanes5-8) and [³⁵S] Methionine labeled proteins bound toGST-NEDD4-WW-Sepharose beads were solublized in sample buffer and wereresolved by SDS-PAGE gel. Equal amounts of [³⁵S]Methionine lysatescorresponding to samples in lanes 1-4 were run on SDS-PAGE gel withoutGST pull-down (Lane 9-12).

[0035]FIG. 7 represents an immunoprecipitation assay. 293 cells wereco-transfected with expression vectors encoding NEDD4-GFP and one offollowing fusion proteins: PMEPA1-V5 (Lane 1), PMEPA1-PY1m-V5 (Lane 2),PMEPA1-PY2m-V5 (Lane 3) or PMEPA1-PY1m/PY2m-V5 (Lane 4). The celllysates from each group were immunoprecipitated with anti-GFP antibodythen subjected to immunoblotting (blot a). Cell lysates from each groupwithout immunoprecipitation were also processed for immunoblotting(blots b and c) to serve as a control. Blots a and b were detected byanti-V5 antibody and blot c was detected by anti-GFP antibody.

[0036]FIG. 8 shows PMEPA1 expression in CWR22 xenograft tumors. Lane 1,sample from CWR22 tumor (androgen dependent). Lanes 2-5, samples from 4individual CWR22R tumors (AR positive but androgen independent).

DETAILED DESCRIPTION OF THE INVENTION

[0037] The present invention provides a method useful in the diagnosisand prognosis of prostate cancer. An aspect of the invention provides amethod to identify ARGs, such as PMEPA1, that exhibit stabletranscriptional induction/repression in response to androgen and havepotential as surrogate markers of the status of the androgen signalingin normal and cancerous epithelial cells of prostate.

[0038] A second aspect of the invention provides for use of theexpression profiles resulting from these methods in diagnostic methods,including, but not limited to, characterizing the treatment response to“hormonal therapy,” correlating expression profiles withclinico-pathologic features, distinguishing indolent prostate cancersfrom those with a more aggressive phenotype, analyzing tumor specimensof patients treated by radical prostate surgery to help defineprognosis, screening candidate genes for the development of apolynucleotide array for use as a blood test for improved prostatecancer detection, and identifying androgen regulated genes that mayserve as biomarkers for response to treatment to screen drugs for thetreatment of advanced prostate cancer.

[0039] As will be readily appreciated by persons having skill in theart, these gene sequences and ESTs described herein can easily besynthesized directly on a support, or presynthesized polynucleotideprobes may be affixed to a support as described, for example, in U.S.Pat. Nos. 5,744,305, 5,837,832, and 5,861,242, each of which isincorporated herein by reference. Furthermore, such arrays may be madein a wide number of variations, combining, probes derived from sequencesidentified by the inventors as up-regulated or down-regulated inresponse to androgen and listed in Table 3 (genes and ESTs derived fromthe inventors' SAGE library that are up-regulated and down-regulated byandrogens) with any of the sequences described in Table 4 (candidategenes and ESTs whose expression are potentially prostate specific orrestricted), Table 5 (previously described genes and ESTs, includingthose associated with androgen signaling, prostate specificity, prostatecancer, and nuclear receptors/regulators with potential interaction withandrogen receptors), Table 6 (genes and ESTs identified from the NIHCGAP database that are differentially expressed in prostate cancer),Table 7 (androgen regulated genes and ESTs derived from the CPDR GenomeSystems ARG Database) and Table 8 (other genes associated with cancers).Tables 3-8 are located at the end of the specification at the end of the“Detailed Description” section and before the “References.” In Table 3,genes in bold type are known androgen-regulated genes based on MedlineSearch. In Table 4, genes in bold type are known prostate-specificgenes.

[0040] Such arrays may be used to detect specific nucleic acid sequencescontained in a target cell or sample, as described in U.S. Pat. Nos.5,744,305, 5,837,832, and 5,861,242, each of which is incorporatedherein by reference. More specifically, in the present invention, thesearrays may be used in methods for the diagnosis or prognosis of prostatecancer, such as by assessing the expression profiles of genes, derivedfrom biological samples such as blood or tissues, that are up-regulatedand down-regulated in response to androgen or otherwise involved inandrogen receptor-mediated signaling. In a preferred embodiment,computer models may be developed for the analysis of expressionprofiles. Moreover, such polynucleotide arrays are useful in methods toscreen drugs for the treatment of advanced prostate cancer. In thesescreening methods, the polynucleotide arrays are used to analyze howdrugs affect the expression of androgen-regulated genes that areinvolved in prostate cancer.

[0041] SAGE analysis. The SAGE technology is based on three mainprinciples: 1) A short sequence tag (10-11 bp) is generated thatcontains sufficient information to identify a transcript, thus, each tagrepresents a signature sequence of a unique transcript; 2) manytranscript tags can be concatenated into a single molecule and thensequenced, revealing the identity of multiple tags simultaneously; 3)quantitation of the number of times a particular tag is observedprovides the expression level of the corresponding transcript (30). Theschematic diagram and the details of SAGE procedure can be obtained fromthe web site: www.genzyme.com/SAGE.

[0042] About fifty percent of SAGE tags identified by the inventorsrepresent ESTs which need to be further analyzed for their proteincoding capacity. The known genes up-regulated or down-regulated byfour-fold (p<0.05) were broadly classified on the basis of thebiochemical functions. SAGE tag defined ARGs were grouped underfollowing categories: transcriptional regulators; RNA processing andtranslation regulators; protein involved in genomic maintenance and cellcycle; protein trafficking/chaperone proteins; energy metabolism,apoptosis and redox regulators; and signal transducers. As determined byPubMed database searches, a majority of genes listed in Table 3 have notbeen described as androgen regulated before. This is the firstcomprehensive list of the functionally defined genes regulated byandrogen in the context of prostatic epithelial cells.

[0043] Although promising candidate ARGs have been identified usingthese approaches, much remains to be learned about the completerepertoire of these genes. SAGE provides both quantitative and highthroughput information with respect to global gene expression profilesof known as well as novel transcripts. We have performed SAGE analysisof the ARGs in the widely studied hormone responsive LNCaP prostatecancer cells treated with and without synthetic androgen, R1881. Ofcourse, this SAGE technique could be repeated with hormones other thanR1881, including other synthetic or natural androgens, such asdihydroxytestosterone, to potentially obtain a slightly different ARGexpression panel. A goal of the inventors was to identify highly inducedand repressed ARGs in LNCaP model which may define a panel of surrogatemarkers for the status androgen signaling in normal as well as cancerousprostate. Here, we report identification and analyses of a comprehensivedatabase of SAGE tags corresponding to well-characterized genes,expressed sequence tags (ESTs) without any protein coding informationand SAGE tags corresponding to novel transcripts. This is the firstreport describing a quantitative evaluation of the global geneexpression profiles of the ARGs in the context of prostatic cancer cellsby SAGE. We have further defined the ARGs on the basis of their knownbiologic/biochemical functions. Our study provides quantitativeinformation on about 23,000 transcripts expressed in LNCaP cells, themost common cell line used in prostate cancer research. Finally,comparison of the LNCaP SAGE tag library and 35 SAGE tag librariesrepresenting diverse cell type/tissues have unraveled a panel of geneswhose expression are prostate specific or prostate abundant. Utilizingthe LNCaP prostate cancer cells, the only well-characterized androgenresponsive prostatic epithelial cells (normal or cancerous), we haveidentified a repertoire of androgen regulated genes by SAGE.

[0044] Utilizing cell-culture systems and cell-signaling agents orexogenous expression of p53 and APC genes, SAGE technology hasidentified novel physiologically relevant transcriptional target geneswhich have unraveled new functions of p53 and APC genes (61-64). Ouranalysis of ARGs has provided identification and quantitative assessmentof induction or repression of a global expression profile of ARGs inLNCaP cells. ARGs resulting from the mutational defects of the AR andthose ARGs unaffected by AR mutations may be identified in this modelsystem. Subsequent androgen regulation analysis of the selected ARGs inAR-positive, primary cultures of normal prostatic epithelial cells, andARGs expression analysis in normal and tumor tissues will clarify normalor abnormal regulation of these ARGs. A panel of highlyinducible/repressible ARGs identified by the inventors may providebio-indicators of the AR transcription factor activity in physiologiccontext. These AR Function Bio-indicators (ARFBs) are useful inassessing the risk of CaP onset and/or progression. Moreover,identification or ARGs may also help in defining the therapeutic targetswhich could lead to effective treatment for hormone refractory cancer,currently a frustrating stage of the disease with limited therapeuticoptions.

[0045] Characterization of a SAGE-defined EST that exhibited the highestlevel of induction in LNCaP cells responding to R1881 led to thediscovery of a novel, androgen-induced gene, PMEPA1, which encodes apolypeptide with a type lb transmembrane domain. A Protein sequencesimilarity search showed homology to C18orf1, a novel gene located onchromosome 18 that is mainly expressed in brain with multipletranscriptional variants (Yoshikawa et al., 1998). In addition to thesequence similarity, PMEPA™ also shares other features with C18orf1,e.g., similar size of the predicted protein and similar transmembranedomain as the P1 isoform of C18orf1. Therefore, it is likely that otherisoforms of PMEPA1 may exist.

[0046] Database searches showed that the PMEPA1 sequence matched togenomic clones RP5-1059L7 and 718J7 which were mapped to chromosome20q13.2-13.33. Gain of 20q has been observed in many cancer types,including prostate, bladder, melanoma, colon, pancreas and breast(Brothman et al., 1990; Richter et al., 1998; Bastian et al., 1998; Komet al., 1999; Mahlamaki et al., 1997; Tanner et al., 1996). Chromosome20q gain was also observed during immortalization and may harbor genesinvolved in bypassing senescence (Jarrard et al., 1999; Cuthill et al.,1999). A differentially expressed gene in hormone refractory CaP, UEV-1,mapped to 20q13.2 (Stubbs et al., 1999). These observations indicatethat one or several genes on chromosome 20q may be involved in prostateor other cancer progression. Although we did not observe increasedexpression of PMEPA™ in primary prostate tumors, increased PMEPA1expression was noted in recurrent cancers of CWR22 xenograft.

[0047] PMEPA1 expression is upregulated by androgens in a time- andconcentration-specific manner in LNCaP cells. This observationunderscores the potential of measuring PMEPA1 expression as one of thesurrogate markers of androgen receptor activity in vivo in theepithelial cells of prostate tissue. Prostate cancer is androgendependent and its growth in prostate is mediated by a network of ARGsthat remains to be fully characterized. Most prostate cancers respond toandrogen withdrawal but relapse after the initial response (Koivisto etal., 1998). The growth of the relapsed tumors is androgen independenteven though tumors are positive for the expression of the AR (Bentel etal., 1996).

[0048] One of the hypotheses of how cancer cells survive and grow in thelow androgen environment is the sensitization or the activation of theAR pathway (Jenster et al., 1999). Studies have shown increasedexpression of the ARGs or amplification of AR in androgen independentprostate cancer tissues (Gregory et al., 1998; Lin et al., 1999). Wehave observed that PMEPA1 was expressed in all CWR22R tumors andincreased expression in three of four compared with CWR22 tumor. Ourdata support the concept that normally AR-dependent pathways remainactivated, despite the absence of androgen in androgen-independentprostate cancer. There are only limited studies that have addressedwhether ARGs play a role in the transition from androgen dependent tumorto androgen independent tumors. The high level of expression only in theprostate gland indicates that PMEPA1 might have important roles relatedto prostate cell biology or physiology. On the basis of homology ofPMEPA1 to C18orf1 it is tempting to suggest that the PMEPA1 may belongto family of proteins involved in the binding of calcium and LDL.

[0049] ARGs, including PMEPA1, can be used as biomarkers of AR functionreadout in the subset of prostate cancers that may involve abrogation ofandrogen signaling. Furthermore, the newly defined ARGs have potentialto identify novel targets in therapy of hormone refractory prostatecancer.

[0050] The nucleic acid molecules encompassed in the invention includethe following PMEPA1 nucleotide sequence:

[0051] ATGGCGGAGC TGGAGTTTGT TCAGATCATC ATCATCGTGG TGGTGATGAT 50

[0052] GGTGATGGTG GTGGTGATCA CGTGCCTGCT GAGCCACTAC AAGCTGTCTG 100

[0053] CACGGTCCTT CATCAGCCGG CACAGCCAGG GGCGGAGGAG AGAAGATGCC 150

[0054] CTGTCCTCAG AAGGATGCCT GTGGCCCTCG GAGAGCACAG TGTCAGGCAA 200

[0055] CGGAATCCCA GAGCCGCAGG TCTACGCCCC GCCTCGGCCC ACCGACCGCC 250

[0056] TGGCCGTGCC GCCCTTCGCC CAGCGGGAGC GCTTCCACCG CTTCCAGCCC 300

[0057] ACCTATCCGT ACCTGCAGCA CGAGATCGAC CTGCCACCCA CCATCTCGCT 350

[0058] GTCAGACGGG GAGGAGCCCC CACCCTACCA GGGCCCCTGC ACCCTCCAGC 400

[0059] TTCGGGACCC CGAGCAGCAG CTGGAACTGA ACCGGGAGTC GGTGCGCGCA 450

[0060] CCCCCAAACA GAACCATCTT CGACAGTGAC CTGATGGATA GTGCCAGGCT 500

[0061] GGGCGGCCCC TGCCCCCCCA GCAGTAACTC GGGCATCAGC GCCACGTGCT 550

[0062] ACGGCAGCGG CGGGCGCATG GAGGGGCCGC CGCCCACCTA CAGCGAGGTC 600

[0063] ATCGGCCACT ACCCGGGGTC CTCCTTCCAG CACCAGCAGA GCAGTGGGCC 650

[0064] GCCCTCCTTG CTGGAGGGGA CCCGGCTCCA CCACACACAC ATCGCGCCCC 700

[0065] TAGAGAGCGC AGCCATCTGG AGCAAAGAGA AGGATAAACA GAAAGGACAC 750

[0066] CCTCTCTAG (SEQ ID NO. 2) 759

[0067] The amino acid sequences of the polypeptides encoded by thePMEPA1 nucleotide sequences of the invention include:

[0068] MAELEFVQII IIVVVMMVMV VVITCLLSHY KLSARSFISR HSQGRRREDA 50

[0069] LSSEGCLWPS ESTVSGNGIP EPQVYAPPRP TDRLAVPPFA QRERFHRFQP 100

[0070] TYPYLQHEID LPPTISLSDG EEPPPYQGPC TLQLRDPEQQ LELNRESVRA 150

[0071] PPNRTIFDSD LMDSARLGGP CPPSSNSGIS ATCYGSGGRM EGPPPTYSEV 200

[0072] IGHYPGSSFQ HQQSSGPPSL LEGTRLHHTH IAPLESAAIW SKEKDKQKGH 250

[0073] PL* (SEQ ID NO. 3) 252

[0074] The discovery of the nucleic acids of the invention enables theconstruction of expression vectors comprising nucleic acid sequencesencoding polypeptides; host cells transfected or transformed with theexpression vectors; isolated and purified biologically activepolypeptides and fragments thereof; the use of the nucleic acids oroligonucleotides thereof as probes to identify nucleic acid encodingproteins having PMEPA I-like activity; the use of single-stranded senseor antisense oligonucleotides from the nucleic acids to inhibitexpression of polynucleotides encoded by the PMEPA1 gene; the use ofsuch polypeptides and fragments thereof to generate antibodies; the useof the antibodies to purify PMEPA1 polypeptides; and the use of thenucleic acids, polypeptides, and antibodies of the invention to detect,prevent, and treat prostate cancer (e.g., prostatic intraepithelialneoplasia (PIN), adenocarcinomas, nodular hyperplasia, and large ductcarcinomas) and prostate-related diseases (e.g., benign prostatichyperplasia).

[0075] As summarized below and explained in further detail in theExamples that follow, our evaluation of PMEPA1 indicates it is aprostate-abundant androgen regulated gene with roles in cell growthcontrol and tumorigenesis. Loss or reduced PMEPA1 expression in prostatecancer correlates with a higher risk or probability of prostatetumorigenesis or progression (e.g., advanced stages of prostate cancer,such as non-organ defined cancer, where tumors extend beyond theprostate gland), particularly after surgery as primary therapy. Thus,alterations in the level, expression, and activity of PMEPA1 and/or itsencoded polypeptide provides useful information about the clinicalbehavior of prostate cancer. Part of our evaluation involved a PMEPA1protein sequence homology search that showed 83% identity to a recentlyreported gene, N4WBP4 (Example 8). N4WBP4 encodes a NEDD4 WW domainbinding protein with two PY motifs that is expressed in mouse embryo[Jolliffe et al., Biochem. J., 351: 557-565, 2000]. The PY motif is aproline-rich peptide sequence with a consensus PPXY sequence (where Xcan be any amino acid) that can bind to proteins with WW domains[Jolliffe et al., Biochem. J., 351: 557-565, 2000; Harvey K et al.,Trends Cell Biol., 9: 166-169, 1999; Hicke L, Cell, 106: 527-530, 2001;Kumar et al., Biochem. Biophys. Res. Commun., 185: 1155-1161, 1992;Kumar et al., Genomics, 40: 435-443, 1997; Sudol M, Trends Biochem.Sci., 21: 161-163, 1996; Harvey et al., J. Biol. Chem., 277: 9307-9317,2002; and Brunschwig et al., Cancer Res., 63: 1568-1575, 2003]. NEDD4was originally identified as a developmentally regulated gene in miceand is a ubiquitin-protein ligase (E3) that is involved in theubiquitin-dependent proteasome-mediated protein degradation pathway.Further studies revealed that NEDD4 is implicated in diverse cellularfunctions, such as regulation of membrane channels and permeases,endocytosis, virus budding, cell cycle, transcription and proteintrafficking [Harvey et al., Trends Cell Biol., 9: 166-169, 1999; HickeL, Cell, 106: 527-530, 2001]. The WW domain present in the NEDD4 proteinis a module with two highly conserved tryptophans that bind to severaltarget proteins containing a PY motif.

[0076] As explained in Example 9, we discovered that PMEPA1 is a NEDD4binding protein and that the binding of PMEPA1 to NEDD4 is mediated bythe PY motifs of PMEPA1. Mutating the PY motifs significantly reducesthe binding of PMEPA1 to NEDD4. In addition, the homology of PMEPA1 tothe NEDD4-binding protein indicates that PMEPA1 may also regulateprotein turnover via ubiquitinylation and proteasome pathways in thecell. This is further supported by our observation that PMEPA1 localizesto the Golgi apparatus (Example 11).

[0077] Further, we recently found that PMEPA1 expression in LNCaP cellsdown regulates androgen receptor protein and modulates the expression ofgenes that are transcriptionally regulated by androgen receptor (Example10). This shows that PMEPA1 functions in androgen receptor regulation.

[0078] Our data also show that PMEPA1 inhibits the growth of prostatecancer cells (Example 12). More specifically, the coding region ofPMEPA1 was inserted into an expression vector and transfected into 293cell (kidney) and LNCaP cells (prostate cancer). Cell proliferation andcell cycle analysis showed that there was no difference between PMEPA1overexpressed 293 cell and control vector transfected 293 cells. HoweverLNCaP cells overexpressing PMEPA1 exhibited significant cell growthinhibition. Similar growth inhibition was observed in other prostatecancer cell lines.

[0079] In addition, in a quantitative evaluation of PMEPA1 expression inprimary prostate cancers, we found that 40 of 62 (64.5%) matchedprostate specimens exhibited decreased expression of PMEPA1 in tumortissues, indicating a correlation between reduced PMEPA1 expression andprostate tumorigenesis (Example 13). When these expression patterns werestratified by organ confined and non-organ confined tumors, a higherpercentage of patients exhibited reduced expression of PMEPA1 innon-organ confined tumor (68%) vs. organ-confined tumor (44%),indicating that reduced PMEPA1 expression correlates with an increasedprobability of advanced prostate cancer.

[0080] Nucleic Acid Molecules

[0081] In a particular embodiment, the invention relates to certainisolated nucleotide sequences that are free from contaminatingendogenous material. A “nucleotide sequence” refers to a polynucleotidemolecule in the form of a separate fragment or as a component of alarger nucleic acid construct. The nucleic acid molecule has beenderived from DNA or RNA isolated at least once in substantially pureform and in a quantity or concentration enabling identification,manipulation, and recovery of its component nucleotide sequences bystandard biochemical methods (such as those outlined in (Sambrook etal., Molecular Cloning: A Laboratory Manual, 3rd ed., Cold Spring HarborLaboratory, Cold Spring Harbor, N.Y. (www.molecularcloning.com). Suchsequences are preferably provided and/or constructed in the form of anopen reading frame uninterrupted by internal non-translated sequences,or introns, that are typically present in eukaryotic genes. Sequences ofnon-translated DNA can be present 5′ or 3′ from an open reading frame,where the same do not interfere with manipulation or expression of thecoding region.

[0082] Nucleic acid molecules of the invention include DNA in bothsingle-stranded and double-stranded form, as well as the RNA complementthereof. DNA includes, for example, cDNA, genomic DNA, chemicallysynthesized DNA, DNA amplified by PCR, and combinations thereof. GenomicDNA may be isolated by conventional techniques, e.g., using the SEQ IDNO: 1 or SEQ ID NO:2, or a suitable fragment thereof, as a probe.

[0083] The DNA molecules of the invention include full length genes aswell as polynucleotides and fragments thereof. The full length gene mayalso include the N-terminal signal peptide. Other embodiments includeDNA encoding a soluble form, e.g., encoding the extracellular domain ofthe protein, either with or without the signal peptide.

[0084] The nucleic acids of the invention are preferentially derivedfrom human sources, but the invention includes those derived fromnon-human species, as well.

[0085] Preferred Sequences

[0086] The particularly preferred nucleotide sequence of the inventionis SEQ ID NO:2, as set forth above. The sequence of amino acids encodedby the DNA of SEQ ID NO:2 is shown in SEQ ID NO:3.

[0087] Additional Sequences

[0088] Due to the known degeneracy of the genetic code, where more thanone codon can encode the same amino acid, a DNA sequence can vary fromthat shown in SEQ ID NO:2, and still encode a polypeptide having theamino acid sequence of SEQ ID NO:3. Such variant DNA sequences canresult from silent mutations (e.g., occurring during PCR amplification),or can be the product of deliberate mutagenesis of a native sequence.

[0089] The invention thus provides isolated DNA sequences encodingpolypeptides of the invention, selected from: (a) DNA comprising thenucleotide sequence of SEQ ID NO:2; (b) DNA encoding the polypeptide ofSEQ ID NO:3; (c) DNA capable of hybridization to a DNA of (a) or (b)under conditions of moderate stringency and which encode polypeptides ofthe invention, wherein the polypeptides inhibit the growth of LNCaPcells in a colony-forming assay; (d) DNA capable of hybridization to aDNA of (a) or (b) under conditions of high stringency and which encodespolypeptides of the invention, wherein the polypeptides inhibit thegrowth of LNCaP cells in a colony-forming assay, and (e) DNA which isdegenerate as a result of the genetic code to a DNA defined in (a), (b),(c), or (d) and which encode polypeptides of the invention. Of course,polypeptides encoded by such DNA sequences are encompassed by theinvention.

[0090] As used herein, conditions of moderate stringency can be readilydetermined by those having ordinary skill in the art based on, forexample, the length of the DNA. The basic conditions are set forth by(Sambrook et al. Molecular Cloning: A Laboratory Manual, 3^(rd) ed.,Cold Spring Harbor Laboratory Press, (www.molecularcloning.com)), andinclude use of a prewashing solution for the nitrocellulose filters5×SSC, 0.5% SDS, 1.0 mM EDTA (pH 8.0), hybridization conditions of about50% formamide, 6×SSC at about 42° C. (or other similar hybridizationsolution, such as Stark's solution, in about 50% formamide at about 42°C.), and washing conditions of about 60° C., 0.5×SSC, 0.1% SDS.Conditions of high stringency can also be readily determined by theskilled artisan based on, for example, the length of the DNA. Generally,such conditions are defined as hybridization conditions as above, andwith washing at approximately 68° C., 0.2×SSC, 0.1% SDS. The skilledartisan will recognize that the temperature and wash solution saltconcentration can be adjusted as necessary according to factors such asthe length of the probe.

[0091] Also included as an embodiment of the invention is DNA encodingpolypeptide fragments and polypeptides comprising inactivatedN-glycosylation site(s), inactivated protease processing site(s), orconservative amino acid substitution(s), as described below.

[0092] In another embodiment, the nucleic acid molecules of theinvention also comprise nucleotide sequences that are at least 80%identical to a native sequence (e.g., SEQ ID NO:2). Also contemplatedare embodiments in which a nucleic acid molecule comprises a sequencethat is at least 90% identical, at least 95% identical, at least 98%identical, at least 99% identical, or at least 99.9% identical to anative sequence (e.g., SEQ ID NO:2).

[0093] The percent identity may be determined by visual inspection andmathematical calculation. Alternatively, the percent identity of twonucleic acid sequences can be determined by comparing sequenceinformation using the GAP computer program, version 6.0 described by(Devereux et al., Nucl. Acids Res., 12:387 (1984)) and available fromthe University of Wisconsin Genetics Computer Group (UWGCG). Thepreferred default parameters for the GAP program include: (1) a unarycomparison matrix (containing a value of 1 for identities and 0 fornon-identities) for nucleotides, and the weighted comparison matrix of(Gribskov and Burgess, Nucl. Acids Res., 14:6745 (1986)), as describedby (Schwartz and Dayhoff, eds., Atlas of Protein Sequence and Structure,National Biomedical Research Foundation, pp. 353-358 (1979)); (2) apenalty of 3.0 for each gap and an additional 0.10 penalty for eachsymbol in each gap; and (3) no penalty for end gaps. Other programs usedby one skilled in the art of sequence comparison may also be used.

[0094] The invention also provides isolated nucleic acids useful in theproduction of polypeptides. Such polypeptides may be prepared by any ofa number of conventional techniques. A DNA sequence encoding a PMEPA1polypeptide, or desired fragment thereof may be subcloned into anexpression vector for production of the polypeptide or fragment. The DNAsequence advantageously is fused to a sequence encoding a suitableleader or signal peptide. Alternatively, the desired fragment may bechemically synthesized using known techniques. DNA fragments also may beproduced by restriction endonuclease digestion of a full length clonedDNA sequence, and isolated by electrophoresis on agarose gels. Ifnecessary, oligonucleotides that reconstruct the 5′ or 3′ terminus to adesired point may be ligated to a DNA fragment generated by restrictionenzyme digestion. Such oligonucleotides may additionally contain arestriction endonuclease cleavage site upstream of the desired codingsequence, and position an initiation codon (ATG) at the N-terminus ofthe coding sequence.

[0095] The well-known polymerase chain reaction (PCR) procedure also maybe used to isolate and amplify a DNA sequence encoding a desired proteinfragment. Oligonucleotides that define the desired termini of the DNAfragment are employed as 5′ and 3′ primers. The oligonucleotides mayadditionally contain recognition sites for restriction endonucleases, tofacilitate insertion of the amplified DNA fragment into an expressionvector. PCR techniques are described in (Saiki et al., Science, 239:487(1988)); (Wu et al., Recombinant DNA Methodology, eds., Academic Press,Inc., San Diego, pp. 189-196 (1989)); and (Innis et al., PCR Protocols:A Guide to Methods and Applications, eds., Academic Press, Inc. (1990)).

[0096] Polypeptides and Fragments Thereof

[0097] The invention encompasses polypeptides and fragments thereof invarious forms, including those that are naturally occurring or producedthrough various techniques such as procedures involving recombinant DNAtechnology. Such forms include, but are not limited to, derivatives,variants, and oligomers, as well as fusion proteins or fragmentsthereof.

[0098] Polypeptides and Fragments Thereof

[0099] The polypeptides of the invention include full length proteinsencoded by the nucleic acid sequences set forth above. Particularlypreferred polypeptides comprise the amino acid sequence of SEQ ID NO:3.

[0100] As discussed in Example 8, SEQ ID NO:3 shares 83% identity to aNEDD4 WW binding protein and contains two PY motifs, i.e., PPPY (SEQ IDNO:80) (“PY1”) and PPTY (SEQ ID NO:81) (“PY2”). The PPXY motif, where Xcan be any amino acid, has been shown to facilitate binding with WWdomain-containing proteins. We demonstrate in the Examples that PMEPA1binds to the NEDD4 protein, which contains WW domains. NEDD4 is aubiquitin-protein ligase (E3) that is involved in theubiquitin-dependent proteasome-mediated protein degradation pathway.

[0101] Assays for determining whether a polypeptide, such as PMEPA1,binds to other proteins having a WW domain are well-known in the art andinclude strategies such as combinatorial peptide libraries, affinitychromatography, expression library screening, and yeast two-hybridscreening (Kay et al. (2000) FEBS Lett., 480:55-62; Frederick et al.(1999) Mol. Cell. Biol., 19: 2330-2337; Dai and Pendergast (1995) GenesDev., 9:2569-2582; Kitamura et al. (1996) Biochem. Biophys. Res.Commun., 219:509-514; Richard et al. (1995) Mol. Cell. Biol. 15:186-197;and Sudol (1994) Oncogene 9:2145-2152).

[0102] The experimental data presented in the Examples show that PMEPA1negatively regulates cancer cell growth. Loss of such function favorstumorigenesis or progression of existing disease. Thus, PMEPA1 maysuppress tumorigenesis or cancer progression by interacting with WWdomain-containing molecules. The homology of PMEPA1 to the NEDD4-bindingprotein and the ability of PMEPA1 to bind NEDD4 indicates that PMEPA1may regulate protein turnover via ubiquitinylation and proteasomepathways in the cell. This mechanism is, of course, merely proposed.Moreover, it is not the only mechanism by which PMEPA1 may exert itsfunction. The present invention is not limited to any particularmechanism of PMEPA1 activity.

[0103] In one embodiment, a polypeptide of the invention comprises anamino acid sequence as set out in SEQ ID NO:3. In another embodiment,the polypeptide comprises an amino acid sequence substantially as setout in SEQ ID NO:3. In yet another embodiment, the polypeptide comprisesan amino acid sequence that is at least 80%, 90%, 95%, 96%, 97%, 98%,99%, OR 99.9% identical to SEQ ID NO:3, and preferably the polypeptideinhibits prostate cancer cell growth, as demonstrated, for example, in acolony-forming assay, such as the one described in Example 12.Inhibiting cell growth refers to a decrease in cell growth in thepresence of a PMEPA1 polypeptide, relative to the cell growth in theabsence of the PMEPA1 polypeptide. Alternatively, if a cell has a basallevel of PMEPA1 polypeptide expression, it refers to a decrease in cellgrowth in the presence of increased levels of PMEPA1 polypeptide,relative to cell growth in the presence of the basal level of PMEPA1polypeptide. Cell growth can be measured using conventional assays, suchas the colony-forming assay described in the examples. As discussed infurther detail below, these polypeptides may be produced by recombinantDNA techniques. Percent identity may be determined by visual inspectionand mathematical calculation. Alternatively, the percent identity of twoprotein sequences can be determined by comparing sequence informationusing the GAP computer program, based on the algorithm of (Needleman andWunsch, J. Mol. Bio., 48:443 (1970)) and available from the Universityof Wisconsin Genetics Computer Group (UWGCG). The preferred defaultparameters for the GAP program include: (1) a scoring matrix, blosum62,as described by (Henikoff and Henikoff Proc. Natl. Acad. Sci. USA,89:10915 (1992)); (2) a gap weight of 12; (3) a gap length weight of 4;and (4) no penalty for end gaps. Other programs used by one skilled inthe art of sequence comparison may also be used.

[0104] The polypeptides of the invention may be membrane bound or theymay be secreted and thus soluble. Soluble polypeptides are capable ofbeing secreted from the cells in which they are expressed. In general,soluble polypeptides may be identified (and distinguished fromnon-soluble membrane-bound counterparts) by separating intact cellswhich express the desired polypeptide from the culture medium, e.g., bycentrifugation, and assaying the medium (supernatant) for the presenceof the desired polypeptide. The presence of polypeptide in the mediumindicates that the polypeptide was secreted from the cells and thus is asoluble form of the protein.

[0105] In one embodiment, the soluble polypeptides and fragments thereofcomprise all or part of the extracellular domain, but lack thetransmembrane region that would cause retention of the polypeptide on acell membrane. A soluble polypeptide may include the cytoplasmic domain,or a portion thereof, as long as the polypeptide is secreted from thecell in which it is produced.

[0106] In general, the use of soluble forms is advantageous for certainapplications. Purification of the polypeptides from recombinant hostcells is facilitated, since the soluble polypeptides are secreted fromthe cells. Further, soluble polypeptides are generally more suitable forintravenous administration.

[0107] The invention also provides polypeptides and fragments of theextracellular domain that retain a desired biological activity. Such afragment may be a soluble polypeptide, as described above.

[0108] Also provided herein are polypeptide fragments comprising atleast 20, or at least 30, contiguous amino acids of the sequence of SEQID NO:3. Fragments derived from the cytoplasmic domain find use instudies of signal transduction, and in regulating cellular processesassociated with transduction of biological signals. Polypeptidefragments also may be employed as immunogens, in generating antibodies.

[0109] Variants

[0110] Naturally occurring variants as well as derived variants of thepolypeptides and fragments are provided herein.

[0111] The variants of the invention include, for example, those thatresult from alternate mRNA splicing events or from proteolytic cleavage.Alternate splicing of mRNA may, for example, yield a truncated butbiologically active protein, such as a naturally occurring soluble formof the protein. Variations attributable to proteolysis include, forexample, differences in the N- or C-termini upon expression in differenttypes of host cells, due to proteolytic removal of one or more terminalamino acids from the protein (generally from 1-5 terminal amino acids).Proteins in which differences in amino acid sequence are attributable togenetic polymorphism (allelic variation among individuals producing theprotein) are also contemplated herein.

[0112] Additional variants within the scope of the invention includepolypeptides that may be modified to create derivatives thereof byforming covalent or aggregative conjugates with other chemical moieties,such as glycosyl groups, lipids, phosphate, acetyl groups and the like.Covalent derivatives may be prepared by linking the chemical moieties tofunctional groups on amino acid side chains or at the N-terminus orC-terminus of a polypeptide. Conjugates comprising diagnostic(detectable) or therapeutic agents attached thereto are contemplatedherein, as discussed in more detail below.

[0113] Other derivatives include covalent or aggregative conjugates ofthe polypeptides with other proteins or polypeptides, such as bysynthesis in recombinant culture as N-terminal or C-terminal fusions.Examples of fusion proteins are discussed below in connection witholigomers. Further, fusion proteins can comprise peptides added tofacilitate purification and identification. Such peptides include, forexample, poly-His or the antigenic identification peptides described inU.S. Pat. No. 5,011,912 and in (Hopp et al., Bio/Technology, 6:1204(1988)). One such peptide is the FLAG® peptide,Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys, (SEQ ID NO:4) which is highly antigenicand provides an epitope reversibly bound by a specific monoclonalantibody, enabling rapid assay and facile purification of expressedrecombinant protein. A murine hybridoma designated 4E11 produces amonoclonal antibody that binds the FLAG® peptide in the presence ofcertain divalent metal cations, as described in U.S. Pat. No. 5,011,912,hereby incorporated by reference. The 4E11 hybridoma cell line has beendeposited with the American Type Culture Collection under accession no.HB 9259. Monoclonal antibodies that bind the FLAG® peptide are availablefrom Eastman Kodak Co., Scientific Imaging Systems Division, New Haven,Conn.

[0114] Among the variant polypeptides provided herein are variants ofnative polypeptides that retain one or more activities associated with afull-length, wild-type, PMEPA1 protein. As one example, such variants oranalogs that have the desired immunogenicity or antigenicity can beused, for example, in immunoassays, for immunization, for inhibition ofPMEPA1 activity, etc. Variants or analogs that retain, or alternativelylack or inhibit, a desired PMEPA1 property of interest can be used asinducers, or inhibitors, respectively, of such property and itsphysiological correlates. These PMEPA1 properties include, but are notlimited to, binding to a WW domain-containing protein or other PMEPA1binding partner, inhibiting cancer cell proliferation, inhibiting theexpression of an androgen receptor, and modulating the expression of agene whose transcription is regulated by the androgen receptor. Bindingaffinity can be measured by conventional procedures, e.g., as describedin U.S. Pat. No. 5,512,457 and as set forth below. Variants or analogsof PMEPA1 can be tested for the desired activity by procedures known inthe art, including but not limited to, the assays described in theExamples.

[0115] In one embodiment, the PMEPA1 variants contain at least onemutation and/or deletion in the at least one of the PY motifs of PMEPA1.These variants can be used, for example, in the treatment ofhypoproliferative disorders. In addition, these variants can be used asimmunogens to generate antibodies.

[0116] Variants include polypeptides that are substantially homologousto the native form, but which have an amino acid sequence different fromthat of the native form because of one or more deletions, insertions orsubstitutions. Particular embodiments include, but are not limited to,polypeptides that comprise from one to ten deletions, insertions orsubstitutions of amino acid residues, when compared to a nativesequence.

[0117] A given amino acid may be replaced, for example, by a residuehaving similar physiochemical characteristics. Examples of suchconservative substitutions include substitution of one aliphatic residuefor another, such as Ile, Val, Leu, or Ala for one another;substitutions of one polar residue for another, such as between Lys andArg, Glu and Asp, or Gln and Asn; or substitutions of one aromaticresidue for another, such as Phe, Trp, or Tyr for one another. Otherconservative substitutions, e.g., involving substitutions of entireregions having similar hydrophobicity characteristics, are well known.

[0118] Similarly, the DNAs of the invention include variants that differfrom a native DNA sequence because of one or more deletions, insertionsor substitutions, but that encode a biologically active polypeptide.

[0119] The invention further includes polypeptides of the invention withor without associated native-pattern glycosylation. Polypeptidesexpressed in yeast or mammalian expression systems (e.g., COS-1 or COS-7cells) can be similar to or significantly different from a nativepolypeptide in molecular weight and glycosylation pattern, dependingupon the choice of expression system. Expression of polypeptides of theinvention in bacterial expression systems, such as E. coli, providesnon-glycosylated molecules. Further, a given preparation may includemultiple differentially glycosylated species of the protein. Glycosylgroups can be removed through conventional methods, in particular thoseutilizing glycopeptidase. In general, glycosylated polypeptides of theinvention can be incubated with a molar excess of glycopeptidase(Boehringer Mannheim).

[0120] Correspondingly, similar DNA constructs that encode variousadditions or substitutions of amino acid residues or sequences, ordeletions of terminal or internal residues or sequences are encompassedby the invention. For example, N-glycosylation sites in the polypeptideextracellular domain can be modified to preclude glycosylation, allowingexpression of a reduced carbohydrate analog in mammalian and yeastexpression systems. N-glycosylation sites in eukaryotic polypeptides arecharacterized by an amino acid triplet Asn-X-Y, wherein X is any aminoacid and Y is Ser or Tbr. Appropriate substitutions, additions, ordeletions to the nucleotide sequence encoding these triplets will resultin prevention of attachment of carbohydrate residues at the Asn sidechain. Alteration of a single nucleotide, chosen so that Asn is replacedby a different amino acid, for example, is sufficient to inactivate anN-glycosylation site. Alternatively, the Ser or Thr can by replaced withanother amino acid, such as Ala. Known procedures for inactivatingN-glycosylation sites in proteins include those described in U.S. Pat.No. 5,071,972 and EP 276,846, hereby incorporated by reference.

[0121] In another example of variants, sequences encoding Cys residuesthat are not essential for biological activity can be altered to causethe Cys residues to be deleted or replaced with other amino acids,preventing formation of incorrect intramolecular disulfide bridges uponfolding or renaturation.

[0122] Other variants are prepared by modification of adjacent dibasicamino acid residues, to enhance expression in yeast systems in whichKEX2 protease activity is present. EP 212,914 discloses the use ofsite-specific mutagenesis to inactivate KEX2 protease processing sitesin a protein. KEX2 protease processing sites are inactivated bydeleting, adding or substituting residues to alter Arg-Arg, Arg-Lys, andLys-Arg pairs to eliminate the occurrence of these adjacent basicresidues. Lys-Lys pairings are considerably less susceptible to KEX2cleavage, and conversion of Arg-Lys or Lys-Arg to Lys-Lys represents aconservative and preferred approach to inactivating KEX2 sites.

[0123] Production of Polypeptides and Fragments Thereof

[0124] Expression, isolation and purification of the polypeptides andfragments of the invention may be accomplished by any suitabletechnique, including but not limited to the following:

[0125] Expression Systems

[0126] The present invention also provides recombinant cloning andexpression vectors containing DNA, as well as host cell containing therecombinant vectors. Expression vectors comprising DNA may be used toprepare the polypeptides or fragments of the invention encoded by theDNA. A method for producing polypeptides comprises culturing host cellstransformed with a recombinant expression vector encoding thepolypeptide, under conditions that promote expression of thepolypeptide, then recovering the expressed polypeptides from theculture. The skilled artisan will recognize that the procedure forpurifying the expressed polypeptides will vary according to such factorsas the type of host cells employed, and whether the polypeptide ismembrane-bound or a soluble form that is secreted from the host cell.

[0127] Any suitable expression system may be employed. The vectorsinclude a DNA encoding a polypeptide or fragment of the invention,operably linked to suitable transcriptional or translational regulatorynucleotide sequences, such as those derived from a mammalian, microbial,viral, or insect gene. Examples of regulatory sequences includetranscriptional promoters, operators, or enhancers, an mRNA ribosomalbinding site, and appropriate sequences which control transcription andtranslation initiation and termination. Nucleotide sequences areoperably linked when the regulatory sequence functionally relates to theDNA sequence. Thus, a promoter nucleotide sequence is operably linked toa DNA sequence if the promoter nucleotide sequence controls thetranscription of the DNA sequence. An origin of replication that confersthe ability to replicate in the desired host cells, and a selection geneby which transformants are identified, are generally incorporated intothe expression vector.

[0128] In addition, a sequence encoding an appropriate signal peptide(native or heterologous) can be incorporated into expression vectors. ADNA sequence for a signal peptide (secretory leader) may be fused inframe to the nucleic acid sequence of the invention so that the DNA isinitially transcribed, and the mRNA translated, into a fusion proteincomprising the signal peptide. A signal peptide that is functional inthe intended host cells promotes extracellular secretion of thepolypeptide. The signal peptide is cleaved from the polypeptide uponsecretion of polypeptide from the cell.

[0129] Suitable host cells for expression of polypeptides includeprokaryotes, yeast or higher eukaryotic cells. Mammalian or insect cellsare generally preferred for use as host cells. Appropriate cloning andexpression vectors for use with bacterial, fungal, yeast, and mammaliancellular hosts are described, for example, in (Pouwels et al. CloningVectors: A Laboratory Manual, Elsevier, New York, (1985)). Cell-freetranslation systems could also be employed to produce polypeptides usingRNAs derived from DNA constructs disclosed herein.

[0130] Prokaryotic Systems

[0131] Prokaryotes include gram-negative or gram-positive organisms.Suitable prokaryotic host cells for transformation include, for example,E. coli, Bacillus subtilis, Salmonella typhimurium, and various otherspecies within the genera Pseudomonas, Streptomyces, and Staphylococcus.In a prokaryotic host cell, such as E. coli, a polypeptide may includean N-terminal methionine residue to facilitate expression of therecombinant polypeptide in the prokaryotic host cell. The N-terminal Metmay be cleaved from the expressed recombinant polypeptide.

[0132] Expression vectors for use in prokaryotic host cells generallycomprise one or more phenotypic selectable marker genes. A phenotypicselectable marker gene is, for example, a gene encoding a protein thatconfers antibiotic resistance or that supplies an autotrophicrequirement. Examples of useful expression vectors for prokaryotic hostcells include those derived from commercially available plasmids such asthe cloning vector pBR322 (ATCC 37017). pBR322 contains genes forampicillin and tetracycline resistance and thus provides simple meansfor identifying transformed cells. An appropriate promoter and a DNAsequence are inserted into the pBR322 vector. Other commerciallyavailable vectors include, for example, pKK223-3 (Pharmacia FineChemicals, Uppsala, Sweden) and pGEM1 (Promega Biotec, Madison, Wis.,USA).

[0133] Promoter sequences commonly used for recombinant prokaryotic hostcell expression vectors include β-lactamase (penicillinase), lactosepromoter system (Chang et al., Nature 275:615 (1978); and (Goeddel etal., Nature 281:544 (1979)), tryptophan (trp) promoter system (Goeddelet al., Nucl. Acids Res. 8:4057 (1980); and EP-A-36776) and tac promoter(Maniatis, Molecular Cloning: A Laboratory Manual, Cold Spring HarborLaboratory, p. 412 (1982)). A particularly useful prokaryotic host cellexpression system employs a phage λP_(L) promoter and a c1857tsthermolabile repressor sequence. Plasmid vectors available from theAmerican Type Culture Collection which incorporate derivatives of theλP_(L) promoter include plasmid pHUB2 (resident in E. coli strain JMB9,ATCC 37092) and pPLc28 (resident in E. coli RR1, ATCC 53082).

[0134] Yeast Systems

[0135] Alternatively, the polypeptides may be expressed in yeast hostcells, preferably from the Saccharomyces genus (e.g., S. cerevisiae).Other genera of yeast, such as Pichia or Kluyveromyces, may also beemployed. Yeast vectors will often contain an origin of replicationsequence from a 2μ yeast plasmid, an autonomously replicating sequence(ARS), a promoter region, sequences for polyadenylation, sequences fortranscription termination, and a selectable marker gene. Suitablepromoter sequences for yeast vectors include, among others, promotersfor metallothionein, 3-phosphoglycerate kinase (Hitzeman et al., J.Biol. Chem. 255:2073 (1980)) or other glycolytic enzymes (Hess et al., JAdv. Enzyme Reg. 7:149 (1968)); and (Holland et al., Biochem. 17:4900(1978)), such as enolase, glyceraldehyde-3-phosphate dehydrogenase,hexokinase, pyruvate decarboxylase, phosphofructokinase,glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvatekinase, triosephosphate isomerase, phospho-glucose isomerase, andglucokinase. Other suitable vectors and promoters for use in yeastexpression are further described in (Hitzeman, EPA-73,657). Anotheralternative is the glucose-repressible ADH2 promoter described by(Russell et al., J. Biol. Chem. 258:2674 (1982)) and (Beier et al.,Nature 300:724 (1982)). Shuttle vectors replicable in both yeast and E.coli may be constructed by inserting DNA sequences from pBR322 forselection and replication in E. coli (Ampr gene and origin ofreplication) into the above-described yeast vectors.

[0136] The yeast α-factor leader sequence may be employed to directsecretion of the polypeptide. The α-factor leader sequence is ofteninserted between the promoter sequence and the structural gene sequence.See, e.g., (Kurjan et al., Cell 30:933 (1982)) and (Bitter et al., Proc.Natl. Acad. Sci. USA 81:5330 (1984)). Other leader sequences suitablefor facilitating secretion of recombinant polypeptides from yeast hostsare known to those of skill in the art. A leader sequence may bemodified near its 3′ end to contain one or more restriction sites. Thiswill facilitate fusion of the leader sequence to the structural gene.

[0137] Yeast transformation protocols are known to those of skill in theart. One such protocol is described by (Hinnen et al., Proc. Natl. Acad.Sci. USA 75:1929 (1978)). The Hinnen et al. protocol selects for Trp⁺transformants in a selective medium, wherein the selective mediumconsists of 0.67% yeast nitrogen base, 0.5% casamino acids, 2% glucose,10 mg/ml adenine and 20 mg/ml uracil.

[0138] Yeast host cells transformed by vectors containing an ADH2promoter sequence may be grown for inducing expression in a “rich”medium. An example of a rich medium is one consisting of 1% yeastextract, 2% peptone, and 1% glucose supplemented with 80 mg/ml adenineand 80 mg/ml uracil. Derepression of the ADH2 promoter occurs whenglucose is exhausted from the medium.

[0139] Mammalian or Insect Systems

[0140] Mammalian or insect host cell culture systems also may beemployed to express recombinant polypeptides. Bacculovirus systems forproduction of heterologous proteins in insect cells are reviewed by(Luckow and Summers, Bio/Technology, 6:47 (1988)). Established celllines of mammalian origin also may be employed. Examples of suitablemammalian host cell lines include the COS-7 line of monkey kidney cells(ATCC CRL 1651) (Gluzman et al., Cell 23:175 (1981)), L cells, C127cells, 3T3 cells (ATCC CCL 163), Chinese hamster ovary (CHO) cells, HeLacells, and BHK (ATCC CRL 10) cell lines, and the CV1/EBNA cell linederived from the African green monkey kidney cell line CV1 (ATCC CCL 70)as described by (McMahan et al., EMBO J, 10: 2821 (1991)).

[0141] Established methods for introducing DNA into mammalian cells havebeen described (Kaufman, R. J., Large Scale Mammalian Cell Culture, pp.15-69 (1990)). Additional protocols using commercially availablereagents, such as Lipofectamine lipid reagent (Gibco/BRL) orLipofectamine-Plus lipid reagent, can be used to transfect cells(Felgner et al., Proc. Natl. Acad. Sci. USA 84:7413-7417 (1987)). Inaddition, electroporation can be used to transfect mammalian cells usingconventional procedures, such as those in (Sambrook et al., MolecularCloning: A Lahoratory Manual, 2 ed. Vol. 1-3, Cold Spring HarborLaboratory Press (1989)). Selection of stable transformants can beperformed using methods known in the art, such as, for example,resistance to cytotoxic drugs. (Kaufman et al., Meth. in Enzymology185:487-511 (1990)), describes several selection schemes, such asdihydrofolate reductase (DHFR) resistance. A suitable host strain forDHFR selection can be CHO strain DX-B 11, which is deficient in DHFR(Urlaub and Chasin, Proc. Natl. Acad. Sci. USA 77:4216-4220 (1980)). Aplasmid expressing the DHFR cDNA can be introduced into strain DX-B 11,and only cells that contain the plasmid can grow in the appropriateselective media. Other examples of selectable markers that can beincorporated into an expression vector include cDNAs conferringresistance to antibiotics, such as G418 and hygromycin B. Cellsharboring the vector can be selected on the basis of resistance to thesecompounds.

[0142] Transcriptional and translational control sequences for mammalianhost cell expression vectors can be excised from viral genomes. Commonlyused promoter sequences and enhancer sequences are derived from polyomavirus, adenovirus 2, simian virus 40 (SV40), and human cytomegalovirus.DNA sequences derived from the SV40 viral genome, for example, SV40origin, early and late promoter, enhancer, splice, and polyadenylationsites can be used to provide other genetic elements for expression of astructural gene sequence in a mammalian host cell. Viral early and latepromoters are particularly useful because both are easily obtained froma viral genome as a fragment, which can also contain a viral origin ofreplication (Fiers et al., Nature 273:113 (1978)); (Kaufman, Meth. inEnzymology (1990)). Smaller or larger SV40 fragments can also be used,provided the approximately 250 bp sequence extending from the Hind IIIsite toward the Bgl I site located in the SV40 viral origin ofreplication site is included.

[0143] Additional control sequences shown to improve expression ofheterologous genes from mammalian expression vectors include suchelements as the expression augmenting sequence element (EASE) derivedfrom CHO cells (Morris et al., Animal Cell Technology, pp. 529-534 andPCT Application WO 97/25420 (1997)) and the tripartite leader (TPL) andVA gene RNAs from Adenovirus 2 (Gingeras et al., J. Biol. Chem.257:13475-13491 (1982)). The internal ribosome entry site (IRES)sequences of viral origin allows dicistronic mRNAs to be translatedefficiently (Oh and Sarnow, Current Opinion in Genetics and Development3:295-300 (1993)); (Ramesh et al., Nucleic Acids Research 24:2697-2700(1996)). Expression of a heterologous cDNA as part of a dicistronic mRNAfollowed by the gene for a selectable marker (e.g. DHFR) has been shownto improve transfectability of the host and expression of theheterologous cDNA (Kaufman, Meth. in Enzymology (1990)). Exemplaryexpression vectors that employ dicistronic mRNAs are pTR-DC/GFPdescribed by (Mosser et al., Biotechniques 22:150-161 (1997)), and p2A5Idescribed by (Morris et al., Animal Cell Technology, pp. 529-534(1997)).

[0144] A useful high expression vector, pCAVNOT, has been described by(Mosley et al., Cell 59:335-348 (1989)). Other expression vectors foruse in mammalian host cells can be constructed as disclosed by (Okayamaand Berg, Mol. Cell. Biol. 3:280 (1983)). A useful system for stablehigh level expression of mammalian cDNAs in C127 murine mammaryepithelial cells can be constructed substantially as described by(Cosman et al., Mol. Immunol. 23:935 (1986)). A useful high expressionvector, PMLSV N1/N4, described by (Cosman et al., Nature 312:768(1984)), has been deposited as ATCC 39890. Additional useful mammalianexpression vectors are described in EP-A-0367566, and in WO 91/18982,incorporated by reference herein. In yet another alternative, thevectors can be derived from retroviruses.

[0145] Another useful expression vector, pFLAG®, can be used. FLAG®technology is centered on the fusion of a low molecular weight (1 kD),hydrophilic, FLAG® marker peptide to the N-terminus of a recombinantprotein expressed by pFLAG® expression vectors. pDC311 is anotherspecialized vector used for expressing proteins in CHO cells. pDC311 ischaracterized by a bicistronic sequence containing the gene of interestand a dihydrofolate reductase (DHFR) gene with an internal ribosomebinding site for DHFR translation, an expression augmenting sequenceelement (EASE), the human CMV promoter, a tripartite leader sequence,and a polyadenylation site.

[0146] Purification

[0147] The invention also includes methods of isolating and purifyingthe polypeptides and fragments thereof.

[0148] Isolation and Purification

[0149] The “isolated” polypeptides or fragments thereof encompassed bythis invention are polypeptides or fragments that are not in anenvironment identical to an environment in which it or they can be foundin nature. The “purified” polypeptides or fragments thereof encompassedby this invention are essentially free of association with otherproteins or polypeptides, for example, as a purification product ofrecombinant expression systems such as those described above or as apurified product from a non-recombinant source such as naturallyoccurring cells and/or tissues.

[0150] In one preferred embodiment, the purification of recombinantpolypeptides or fragments can be accomplished using fusions ofpolypeptides or fragments of the invention to another polypeptide to aidin the purification of polypeptides or fragments of the invention.

[0151] With respect to any type of host cell, as is known to the skilledartisan, procedures for purifying a recombinant polypeptide or fragmentwill vary according to such factors as the type of host cells employedand whether or not the recombinant polypeptide or fragment is secretedinto the culture medium.

[0152] In general, the recombinant polypeptide or fragment can beisolated from the host cells if not secreted, or from the medium orsupernatant if soluble and secreted, followed by one or moreconcentration, salting-out, ion exchange, hydrophobic interaction,affinity purification or size exclusion chromatography steps. As tospecific ways to accomplish these steps, the culture medium first can beconcentrated using a commercially available protein concentrationfilter, for example, an Amicon or Millipore Pellicon ultrafiltrationunit. Following the concentration step, the concentrate can be appliedto a purification matrix such as a gel filtration medium. Alternatively,an anion exchange resin can be employed, for example, a matrix orsubstrate having pendant diethylaminoethyl (DEAE) groups. The matricescan be acrylamide, agarose, dextran, cellulose or other types commonlyemployed in protein purification. Alternatively, a cation exchange stepcan be employed. Suitable cation exchangers include various insolublematrices comprising sulfopropyl or carboxymethyl groups. In addition, achromatofocusing step can be employed. Alternatively, a hydrophobicinteraction chromatography step can be employed. Suitable matrices canbe phenyl or octyl moieties bound to resins. In addition, affinitychromatography with a matrix which selectively binds the recombinantprotein can be employed. Examples of such resins employed are lectincolumns, dye columns, and metal-chelating columns. Finally, one or morereversed-phase high performance liquid chromatography (RP-HPLC) stepsemploying hydrophobic RP-HPLC media, (e.g., silica gel or polymer resinhaving pendant methyl, octyl, octyldecyl or other aliphatic groups) canbe employed to further purify the polypeptides. Some or all of theforegoing purification steps, in various combinations, are well knownand can be employed to provide an isolated and purified recombinantprotein.

[0153] It is also possible to utilize an affinity column comprising apolypeptide-binding protein of the invention, such as a monoclonalantibody generated against polypeptides of the invention, toaffinity-purify expressed polypeptides. These polypeptides can beremoved from an affinity column using conventional techniques, e.g., ina high salt elution buffer and then dialyzed into a lower salt bufferfor use or by changing pH or other components depending on the affinitymatrix utilized, or be competitively removed using the naturallyoccurring substrate of the affinity moiety, such as a polypeptidederived from the invention.

[0154] In this aspect of the invention, polypeptide-binding proteins,such as the anti-polypeptide antibodies of the invention or otherproteins that may interact with the polypeptide of the invention, can bebound to a solid phase support such as a column chromatography matrix ora similar substrate suitable for identifying, separating, or purifyingcells that express polypeptides of the invention on their surface.Adherence of polypeptide-binding proteins of the invention to a solidphase contacting surface can be accomplished by any means, for example,magnetic microspheres can be coated with these polypeptide-bindingproteins and held in the incubation vessel through a magnetic field.Suspensions of cell mixtures are contacted with the solid phase that hassuch polypeptide-binding proteins thereon. Cells having polypeptides ofthe invention on their surface bind to the fixed polypeptide-bindingprotein and unbound cells then are washed away. This affinity-bindingmethod is useful for purifying, screening, or separating suchpolypeptide-expressing cells from solution. Methods of releasingpositively selected cells from the solid phase are known in the art andencompass, for example, the use of enzymes. Such enzymes are preferablynon-toxic and non-injurious to the cells and are preferably directed tocleaving the cell-surface binding partner.

[0155] Alternatively, mixtures of cells suspected of containingpolypeptide-expressing cells of the invention first can be incubatedwith a biotinylated polypeptide-binding protein of the invention.Incubation periods are typically at least one hour in duration to ensuresufficient binding to polypeptides of the invention. The resultingmixture then is passed through a column packed with avidin-coated beads,whereby the high affinity of biotin for avidin provides the binding ofthe polypeptide-binding cells to the beads. Use of avidin-coated beadsis known in the art. See (Berenson, et al. J. Cell. Biochem., 10D:239(1986)). Wash of unbound material and the release of the bound cells isperformed using conventional methods.

[0156] The desired degree of purity depends on the intended use of theprotein. A relatively high degree of purity is desired when thepolypeptide is to be administered in vivo, for example. In such a case,the polypeptides are purified such that no protein bands correspondingto other proteins are detectable upon analysis by SDS-polyacrylamide gelelectrophoresis (SDS-PAGE). It will be recognized by one skilled in thepertinent field that multiple bands corresponding to the polypeptide maybe visualized by SDS-PAGE, due to differential glycosylation,differential post-translational processing, and the like. Mostpreferably, the polypeptide of the invention is purified to substantialhomogeneity, as indicated by a single protein band upon analysis bySDS-PAGE. The protein band may be visualized by silver staining,Coomassie blue staining, or (if the protein is radiolabeled) byautoradiography.

[0157] Production of Antibodies

[0158] Antibodies that are immunoreactive with the polypeptides of theinvention are provided herein. Such antibodies specifically bind to thepolypeptides via the antigen-binding sites of the antibody (as opposedto non-specific binding). Thus, the polypeptides, fragments, variants,fusion proteins, etc., as set forth above may be employed as“immunogens” in producing antibodies immunoreactive therewith. Morespecifically, the polypeptides, fragment, variants, fusion proteins,etc. contain antigenic determinants or epitopes that elicit theformation of antibodies.

[0159] These antigenic determinants or epitopes can be either linear orconformational (discontinuous). Linear epitopes are composed of a singlesection of amino acids of the polypeptide, while conformational ordiscontinuous epitopes are composed of amino acids sections fromdifferent regions of the polypeptide chain that are brought into closeproximity upon protein folding (C. A. Janeway, Jr. and P. Travers,Immuno Biology 3:9, Garland Publishing Inc., 2nd ed. (1996)). Becausefolded proteins have complex surfaces, the number of epitopes availableis quite numerous; however, due to the conformation of the protein andsteric hinderances, the number of antibodies that actually bind to theepitopes is less than the number of available epitopes (C. A. Janeway,Jr. and P. Travers, Immuno Biology 2:14, Garland Publishing Inc., 2nded. (1996)). Epitopes may be identified by any of the methods known inthe art.

[0160] Thus, one aspect of the present invention relates to theantigenic epitopes of the polypeptides of the invention. Such epitopesare useful for raising antibodies, in particular monoclonal antibodies,as described in more detail below. Additionally, epitopes from thepolypeptides of the invention can be used as research reagents, inassays, and to purify specific binding antibodies from substances suchas polyclonal sera or supernatants from cultured hybridomas. Suchepitopes or variants thereof can be produced using techniques well knownin the art such as solid-phase synthesis, chemical or enzymatic cleavageof a polypeptide, or using recombinant DNA technology.

[0161] As to the antibodies that can be elicited by the epitopes of thepolypeptides of the invention, whether the epitopes have been isolatedor remain part of the polypeptides, both polyclonal and monoclonalantibodies may be prepared by conventional techniques. See, for example,(Kennet et al., Monoclonal Antibodies, Hybridomas: A New Dimension inBiological Analyses, eds., Plenum Press, New York (1980); and Harlow andLand, Antibodies: A Laboratory Manual, eds., Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y., (1988)).

[0162] Hybridoma cell lines that produce monoclonal antibodies specificfor the polypeptides of the invention are also contemplated herein. Suchhybridomas may be produced and identified by conventional techniques.One method for producing such a hybridoma cell line comprises immunizingan animal with a polypeptide; harvesting spleen cells from the immunizedanimal; fusing said spleen cells to a myeloma cell line, therebygenerating hybridoma cells; and identifying a hybridoma cell line thatproduces a monoclonal antibody that binds the polypeptide. Themonoclonal antibodies may be recovered by conventional techniques.

[0163] The monoclonal antibodies of the present invention includechimeric antibodies, e.g., humanized versions of murine monoclonalantibodies. Such humanized antibodies may be prepared by knowntechniques and offer the advantage of reduced immunogenicity when theantibodies are administered to humans. In one embodiment, a humanizedmonoclonal antibody comprises the variable region of a murine antibody(or just the antigen binding site thereof) and a constant region derivedfrom a human antibody. Alternatively, a humanized antibody fragment maycomprise the antigen binding site of a murine monoclonal antibody and avariable region fragment (lacking the antigen-binding site) derived froma human antibody. Procedures for the production of chimeric and furtherengineered monoclonal antibodies include those described in (Riechmannet al., Nature 332:323 (1988), Liu et al., PNAS 84:3439 (1987), Larricket al., Bio/Technology 7:934 (1989), and Winter and Harris, TIPS 14:139(May 1993)). Procedures to generate antibodies transgenically can befound in GB 2,272,440, U.S. Pat. Nos. 5,569,825 and 5,545,806 andrelated patents claiming priority therefrom, all of which areincorporated by reference herein.

[0164] Antigen-binding fragments of the antibodies, which may beproduced by conventional techniques, are also encompassed by the presentinvention. Examples of such fragments include, but are not limited to,Fab and F(ab′)₂ fragments. Antibody fragments and derivatives producedby genetic engineering techniques are also provided.

[0165] In one embodiment, the antibodies are specific for thepolypeptides of the present invention and do not cross-react with otherproteins. Screening procedures by which such antibodies may beidentified are well known, and may involve immunoaffinitychromatography, for example.

[0166] The following examples further illustrate preferred aspects ofthe invention.

EXAMPLE 1

[0167] Cell Culture and Androgen Stimulation

[0168] LNCaP cells (American Type Culture Collection, Rockville, Md.)were used for SAGE analysis of ARGs. LNCaP cells were maintained in RPMI1640 (Life Technologies, Inc., Gaithersburg, Md.) supplemented with 10%fetal bovine serum (FBS, Life Technologies, Inc., Gaithersburg, Md.) andexperiments were performed on cells between passages 20 and 30. For thestudies of androgen regulation, charcoal/dextran stripped androgen-freeFBS (cFBS, Gemini Bio-Products, Inc., Calabasas, Calif.) was used. LNCaPcells were cultured first in RPMI 1640 with 10% cFBS for 5 days and thenstimulated with 10-8 M of non-metabolizable androgen analog, R1881(DUPONT, Boston, Mass.) for 24 hours. LNCaP cells identically treatedbut without R1881 treatment served as control. Cells were harvested atindicated time and polyA+ RNA was double-selected with Fast Track kit(Invitrogene). The quality of polyA+ was checked by Northernhybridization analysis.

EXAMPLE 2

[0169] SAGE Analysis

[0170] Two SAGE libraries (library LNCaP-C and library LNCaP-T) weregenerated according to the procedure described previously Velculescu etal. (30). Briefly, biotinylated oligo dT primed cDNA was prepared fromfive micrograms of polyA+ RNA from R1881 treated and control LNCaP cellsand biotinylated cDNA was captured on strepravidin coated magnetic beads(Dynal Corporation, MI). cDNA bound to the magnetic beads were digestedby NlaIII followed by ligation to synthetic linkers containing a sitefor anchoring enzyme, NlaIII and a site for tagging enzyme BsmF1. Therestriction digestion of ligated products with BsmF1 resulted in thecapture of 10-11 bp sequences termed as “tags” representing signaturesequence of unique cDNAs. A multi-step strategy combining ligation, PCR,enzymatic digestion and gel purification yielded two tags linkedtogether termed as “ditags.” Ditags were concatamerized, purified andcloned in plasmid pZero cloning vector (Invitrogen, Calif.). The clonescontaining concatamers were screened by PCR and sequenced. The sequenceand the occurrence of each of the SAGE tags was determined using theSAGE software kindly provided by Dr. Kenneth W. Kinzler (Johns HopkinsUniversity School of Medicine, Baltimore, Md.). All the SAGE tagssequences were analyzed for identity to DNA sequence in GenBank(National Center for Biotechnology Information, Bethesda, Md., USA). Therelative abundance of each transcript was determined by dividing thenumber of individual tags by total tags in the library. The copy numberof each gene was calculated assuming there are approximately 300,000transcripts in a cell (Zhang et al., 1997). The differentially expressedSAGE tags were determined by comparing the frequency of occurrence ofindividual tags in the two libraries obtained from the control (libraryLNCaP-C) and R1881 treated LNCaP cells (library LNCaP-T). The resultswere analyzed with t test, and p<0.05 was considered as a statisticallysignificant difference for a specific tag between these two libraries.

EXAMPLE 3

[0171] Kinetics of Androgen Regulation ARGs Defined by SAGE Analysis

[0172] LNCaP cells were cultured in RPMI 1640 with 10% cFBS for 5 days,then stimulated with R1881 at 10-10, 10-8, and 10-6 M for 1, 3, 12, 24,72, 120, 168, and 216 hours. LNCaP cells identically treated but withoutR1881 served as control. The cells were harvested at indicated time andpolyA+ RNA was prepared as described as above. The polyA+ RNA wasfractionated (2 μg/lane) by running through 1% formaldehyde-agarose geland transferred to nylon membrane. The cDNA probes of several ARGs werelabeled with ³²P-dCTP by random priming (Stratagene Cloning Systems, LaJolla, Calif.). The nylon membranes were prehybridized for 2 hrs inhybridization buffer (10 mM Tris-HCl, pH 7.5, 10% Dextran sulfate, 40%Formamide, 5×SSC, 5× Denhardt's solution and 0.25 mg/ml salmon spermDNA) and hybridized to the ³²P labeled probes (1×10⁶ cpm/ml) in the samebuffer at 40° C. for 12-16 hrs. Blots were washed twice in 2×SSC/0.1%SDS for 20 min at room temperature followed by two high-stringency washwith 0.1×SSC/0.1% SDS at 50° C. for 20 min. Nylon membranes were exposedto X-ray film for autoradiography.

EXAMPLE 4

[0173] ARGs Expression Pattern in Cwr22 Model.

[0174] CWR22 (androgen dependent) and CWR22R (androgen relapsed) tumorspecimens were kindly provided by Dr. Thomas Pretlow (Case WesternReserve University School of Medicine). The tissue samples werehomogenized and polyA+ RNA was extracted with Fast Track kit(Invitrogen) following manufacture's protocol. Northern blots wereprepared as described in Example 3 and were hybridized with ³²P labeledprobes of the cDNA of interest.

[0175] Analysis of SAGE tag libraries from R1881 treated LNCaP cells.LNCaP cells were maintained in androgen deprived growth media for fivedays and were treated with synthetic androgen R1881 (10 nm) for 24hours. Since a goal of the inventors was to identify androgen signalingread-out transcripts, we chose conditions of R1881 treatment of LNCaPcells showing a robust and stable transcriptional induction ofwell-characterized prostate-specific androgen regulated genes,prostate-specific antigen (PSA) and NKX3.1 genes. A total of 90,236 tagswere derived from the two SAGE libraries. Of 90,236 tags, 6,757 tagscorresponded to linker sequences, and were excluded from furtheranalysis. The remaining 83,489 tags represented a total of 23,448 knowngenes or ESTs and 1,655 tags did not show any match in the GeneBank database. The relative abundance of the SAGE tags varied between 0.0011% and1.7%. Assuming that there are 18,000 transcripts per cell type and thereare about 83,489 anticipated total transcripts, the estimated abundanceof transcripts will be 0.2-308 copies per cell. This calculationindicated that single copy genes had high chance to be recognized bySAGE analysis in this study. The distribution of transcripts by copynumber suggests that the majority (above 90%) of the genes in ouranalysis are expressed at 1 or 2 copies level/cell. A total of 46,186and 45,309 tags were analyzed in the control (C) and R1881 (T) groupsrespectively. Unique SAGE tags corresponding to known genes, expressedsequence tags (ESTs) and novel transcripts were 15,593 and 15,920 in thecontrol and androgen treated groups respectively. About 94% of theunique SAGE tags in each group showed a match to a sequence in the genebank and 6% SAGE tags represented novel transcripts. The most abundantSAGE tags in both control and androgen treated LNCaP cells representedproteins involved in cellular translation machinery e.g., ribosomalproteins, translation regulators, mitochondrial proteins involved inbio-energetic pathways.

EXAMPLE 5

[0176] Analysis of the ARGs Defined by SAGE Tags

[0177] Of about 15,000 unique tags a total of 136 SAGE tags weresignificantly up-regulated in response to R1881 whereas 215 SAGE tagswere significantly down-regulated (p<0.05). It is important to note thatof 15,000 expressed sequences only 1.5% were androgen responsivesuggesting that expression of only a small subset of genes are regulatedby androgen under our experimental conditions. The ARGs identified bythe inventors are anticipated to represent a hierarchy, where a fractionof ARGs are directly regulated by androgens and others represent theconsequence of the activation of direct down-stream target genes of theAR. Comparison of SAGE tags between control and R1881 also revealed that74 SAGE tags were significantly up-regulated (p<0.05) by four-fold and120 SAGE tags were significantly (p<0.05) down-regulated. Two SAGE tagscorresponding to the PSA gene sequence exhibited highest induction (16fold) between androgen treated (T) and control (C) groups. Anotherprostate specific androgen regulated gene, NKX3.1 was amongsignificantly up-regulated ARGs (8 fold). Prostate specific membraneantigen (PSMA) and Clusterin known to be down-regulated by androgenswere among the SAGE tags exhibiting decreased expression in response toandrogen (PSMA, 4 fold; Clusterin, fold). Therefore, identification ofwell characterized up-regulated and down-regulated ARGs defined by SAGEtags validates the use of LNCaP experimental model for definingphysiologically relevant ARGs in the context of prostatic epithelialcells. It is important to note that about 90% of up-regulated ARGs and98% of the down-regulated ARGs defined by our SAGE analysis were notknown to be androgen-regulated before.

EXAMPLE 6

[0178] Identification of Prostate Specific/Abundant Genes

[0179] LNCaP C/T-SAGE tag libraries were compared to a bank of 35 SAGEtag libraries (http://www.ncbi.nlm.nih.gov/SAGE/) containing 1.5 milliontags from diverse tissues and cell types. Our analysis revealed thatknown prostate specific genes e.g., PSA and NKX3.1 were found only inLNCaP SAGE tag libraries (this report and one LNCaP SAGE library presentin the SAGE tag bank). We have extended this observation to the othercandidate genes and transcripts. On the basis of abundant/uniqueexpression of the SAGE tag defined transcripts in LNCaP SAGE taglibraries relative to other libraries, we have now identified severalcandidate genes and ESTs whose expression are potentially prostatespecific or restricted (Table 4). The utility of such prostate-specificgenes includes: (a) the diagnosis and prognosis of CaP (b) tissuespecific targeting of therapeutic genes (c) candidates for immunotherapyand (d) defining prostate specific biologic functions.

[0180] Genes with defined functions showing at least five fold up ordown-regulation (p<0.05) were broadly classified on the basis of theirbiochemical function, since our results of Northern analysis ofrepresentative SAGE derived ARGs at 5-fold difference showed mostreproducible results. Table 9, presented at the end of thisspecification immediately preceding the “References” section, representsthe quantitative expression profiles of a panel of functionally definedARGs in the context of LNCaP prostate cancer cells. ARGs in thetranscription factor category include proteins involved in the generaltranscription machinery e.g., KAP1/TIF β, CHD4 and SMRT (Douarin et al.,1998; Xu et al., 1999) have been shown to participate in transcriptionalrepression. The mitochondrial transcription factor 1 (mtTF1) was inducedby 8 fold in response to R1881. A recent report describes that anothermember of the nuclear receptor superfamily, the thyroid hormonereceptor, also up-regulates a mitochondrial transcription factorexpression through a specific co-activator, PGC-1 (Wu et al., 1999). Asshown in Table 9 a thyroid hormone receptor related gene, ear-2(Miyajima et al., 1998) was also upregulated by R1881. It is striking tonote that expression of four [NKX3.1 (He et al., 1997), HOX B 13(Sreenath et al., 1999), mtTF1 and PDEF (Oettgen et al., 2000)] of theeight transcription regulators listed in Table 9 appear to be prostatetissue abundant/specific based on published reports as well as ouranalysis described above.

[0181] ARGs also include a number of proteins involved in cellularenergy metabolism and it is possible that some of these enzymes may betranscriptionally regulated by mtTF1. Components of enzymes involved inoxidative decaboxylation: dihydrolipoamide succinyl transferase (Patelet al., 1995), puruvate dehydrogenase E-1 subunit (Ho et al., 1989), andthe electron tansport chain: NADH dehydrogenase 1 beta subcomplex 10(Ton et al., 1997) were upregulated by androgen. VDAC-2 (Blachly-Dysonet al., 1994), a member of small pore forming proteins of the outermitochondrial membrane and which may regulate the transport of smallmetabolites necessary for oxidative-phosphorylation, was also upregulated by androgen. Diazepam binding protein (DBI), a previousreported ARG (Swinnen et al., 1996), known to be associated with theVDAC complex and implicated in a multitude of functions includingmodulation of pheripheral benzodiaepine receptor, acyl-CoA metabolismand mitochondrial steroidogenesis (Knudsen et al., 1993) were alsoinduced by androgen in our study. A thioredoxin like protein(Miranda-Vizuete et al., 1998) which may function in modulating thecellular redox state was down regulated by androgen. In general, itappears that modulation of ARGs involved in regulating cellular redoxstatus and energy metabolism may effect reactive oxygen speciesconcentrations.

[0182] A number of cell proliferation associated proteins regulatingcell cycle, signal transduction and cellular protein trafficking wereupregulated by androgen, further supporting the role of androgens insurvival and growth of prostatic epithelial cells. Androgen regulationof two proteins: XRCC2 (Cartwright et al., 1998) and RPA3 (Umbricht etal., 1993) involved in DNA repair and recombination is a novel andinteresting finding. Induction of these genes may represent a responseto DNA damage due to androgen mediated pro-oxidant shift, or these genessimply represent components of genomic surveillance mechanismsstimulated by cell proliferation. The androgen induction of a p53inducible gene, PIG 8 (Umbricht et al., 1997), is another intriguingfinding as the mouse homolog of this gene, ei24 (Gu et al., 2000), isinduced by etoposide known to generate reactive oxygen species. Inaddition, components of protein kinases modulated by adenyl cyclase,guanyl cyclase and calmodulin involved in various cellular signaltransduction stimuli were also regulated by androgen.

[0183] Gene expression modulations in RNA processing and translationcomponents is consistent with increased protein synthesis expected incells that are switched to a highly proliferative state. Of note isnucleolin, one of the highly androgen induced genes (12 fold) which isan abundant nucleolar protein associating with cell proliferation andplays a direct role in the biogenesis, processing and transport ofribosomes to the cytoplasm (Srivastava and Pollard, 1999). Anotherandrogen up-regulated gene, exportin, a component of the nuclear pore,may be involved in the shuttling of nucleolin. Androgen regulation ofSiahBP1 (Page-McCaw et al., 1999), GRSF-1 (Qian and Wilusz, 1994) andPAIP1 (Craig et al., 1998) suggests a role of androgen signaling in theprocessing of newly transcribed RNAs. Two splicesosomal genes, snRNP-Gand snRNP-E coding for small ribo-nucleoproteins were down-regulated byandrogen. The unr-interacting protein, UNRIP (Hunt et al., 1999) whichis involved in the direct ribosome entry of many viral and some cellularmRNAs into the translational pathway, was the most down-regulated genein response to androgen.

[0184] Quantitative evaluation of gene expression profiles by SAGEapproach have defined yeast transcriptome (Velculescu et al., 1997) andhave identified critical genes in biochemical pathways regulated by p53(Polyak et al., 1997), x-irradiation (Hermeking et al., 1997) and theAPC gene (Korinek et al., 1997). Potential tumor biomarkers in colon(Zhang et al., 1997), lung (Hibi et al., 1998), and breast (Nacht etal., 1999) cancers and genes regulated by other cellular stimuli (Waardet al., 1999; Berg et al., 1999) have also been identified by SAGE. SAGEtechnology has enabled us to develop the first quantitative database ofandrogen regulated transcripts. Comparison of our SAGE tag libraries tothe SAGE TagBank has also revealed a number of new candidate genes andESTs whose expression is potentially abundant or specific to theprostate. We have also identified a large number of transcripts notpreviously defined as ARGs.

[0185] A great majority of functionally defined genes that weremodulated by androgen in our experimental system appear to promote cellproliferation, cell survival, gain of energy and increased oxidativereactions shift in the cells. However, a substantial fraction of theseARGs appears to be androgen specific since they do not exhibitappreciable change in their expression in other studies examining cellproliferation associated genes (Iyer et al., 1999,genome-www.stanford.edu/serum) or estrogen regulated genes in MCF7 cells(Charpentier et al., 2000). The interesting experimental observation ofRipple et al. (Ripple et al., 1997) showing a prooxidant-antioxidantshift induced by androgen in prostate cancer cells is supported by ouridentification of specific ARGs (upregulation of enzymes involved inoxidative reactions, electron transport chain and lipid metabolism inmitochondria and down regulation of thioredoxin like protein) that maybe involved in the induction of oxidative stress by androgen.

EXAMPLE 7

[0186] Characterization of the Androgen-Regulated Gene PMEPA1

[0187] cDNA library screening and Sequencing of cDNA clone. One of theSAGE tags (14 bp) showing the highest induction by androgen (29-fold)exhibited homology to an EST in the GenBank EST database. PCR primers(5′GGCAGAACACTCCGCGCTTCTTAG3′ (SEQ ID NO. 5) and5′CAAGCTCTCTTAGCTTGTGCATTC3′ (SEQ ID NO. 6)) were designed based on theEST sequence (accession number AA310984). RT-PCR was performed using RNAfrom R1881 treated LNCaP cells and the co-identity of the PCR product tothe EST was confirmed by DNA sequencing. Using the PCR product as probe,the normal prostate cDNA library was screened through the serviceprovided by Genome Systems (St. Louis, Mo.). An isolated clone, GS 22381was sequenced using the 310 Genetic Analyzer (PE Applied Biosystems,Foster Calif.) and 750 bp of DNA sequence was defined, which included2/3 of the coding region of PMEPA1. A GenBank search with PMEPA1 cDNAsequence revealed one EST clone (accession number AA088767) homologousto the 5′ region of the PMEPA1 sequence. PCR primers were designed usingthe EST clone (5′ primer) and PMEPA1 (3′ primer) sequence. cDNA fromLNCaP cells was PCR amplified and the PCR product was sequenced usingthe PCR primers. The sequences were verified using at least twodifferent primers. A contiguous sequence of 1,141 bp was generated bythese methods.

[0188] Kinetics of Androgen Regulation of PMEPA1 Expression in LNCaPCells.

[0189] LNCaP cells (American Type Culture Collection, ATCC, RockvilleMd.) were maintained in RPMI 1640 media (Life Technologies, Inc.,Gaithersburg, Md.) supplemented with 10% fetal bovine serum (FBS, LifeTechnologies, Inc., Gaithersburg, Md.) and experiments were performed oncells cultured between passages 20 and 30. For the studies of androgenregulation, charcoal/dextran stripped androgen-free FBS (cFBS, GeminiBio-Products, Inc., Calabasas, Calif.) was used. LNCaP cells werecultured first in RPMI 1640 with 10% cFBS for 5 days, and thenstimulated with R1881 (DUPONT, Boston, Mass.) at 10⁻¹⁰ M and 10⁻⁸ M for3, 6, 12 and 24 hours. LNCaP cells identically treated but without R1881served as control. To study the effects of androgen withdrawal on PMEPA1gene expression, LNCaP cells were cultured in RPMI 1640 with 10% cFBSfor 24, 72 and 96 hours. Poly A+ RNA samples derived from cells treatedwith or without R1881 were extracted at indicated time points with aFast Track mRNA extraction kit (Invitrogen, Carlsbad, Calif.) followingthe manufacturer's protocol. Poly A+ RNA specimens (2 zg/lane) wereelectrophoresed in a 1% formaldehyde-agarose gel and transferred to anylon membrane. Two PMEPA1 probes used for Northern blots analysis were(a) cDNA probe spanning nucleotides 3-437 of PMEPA1 cDNA sequence (SeeTable 1) and (b) 71-mer oligonucleotide between nucleotides 971 to 1,041of PMEPA1 cDNA sequence (See Table 1).

[0190] The cDNA probe was generated by RT-PCR with primers5′CTTGGGTTCGGGTGAAAGCGCC 3′ (SEQ ID NO. 7) (sense) and5′GGTGGGTGGCAGGTCGATCTCG 3′ (SEQ ID NO. 8) (antisense). PMEPA1oligonucleotide and cDNA probes and glyceraldehyde phosphatedehydrogenase gene (GAPDH) cDNA probe were labeled with ³²P-dCTP using3′ end tailing for oligonucleotides (Promega, Madison, Wis.) and randompriming for cDNA (Stratagene, La Jolla, Calif.). The nylon membraneswere treated with hybridization buffer (10 mM Tris-HCl, pH 7.5, 10%Dextran sulfate, 40% Formamide, 5×SSC, 5× Denhardt's solution and 0.25mg/ml salmon sperm DNA) for two hours followed by hybridization in thesame buffer containing the ³²P labeled probes (1×10⁶ cpm/ml) for 12-16hrs at 40° C. Blots were washed twice in 2×SSC/0.1% SDS for 20 min atroom temperature followed by two high-stringency washes with0.1×SSC/0.1% SDS at 58° C. for 20 min. Nylon membranes were exposed toX-ray film for autoradiography. The bands on films were then quantifiedwith NIH-Image processing software.

[0191] PMEPA1 expression analysis in CWR22 tumors. CWR22 is anandrogen-dependent, serially transplantable nude mouse xenograft derivedfrom a primary human prostate cancer. Transplanted CWR22 tumors arepositive for AR and the growth of CWR22 is androgen dependent. CWR22tumors regress initially upon castration followed by a relapse. Therecurrent CWR22 tumors (CWR22R) express AR, but the growth of thesetumors become androgen-independent (Gregory et al., 1998; Nagabhushan etal., 1996). One CWR22 and four CWR22R tumor specimens were kindlyprovided by Dr. Thomas Pretlow's laboratory (Case Western ReserveUniversity School of Medicine). Tumor tissues were homogenized and polyA+ RNA were extracted as above. PolyA+ RNA blots were made andhybridized as described above.

[0192] PMEPA1 expression analysis in multiple human tissues and celllines. Multiple Tissue Northern blots containing mRNA samples from 23human tissues and Master Dot blots containing mRNA samples from 50different human tissues were purchased from ClonTech (Palo Alto,Calif.). The blots were hybridized with PMEPA1 cDNA and oligo probes, asdescribed above. The expression of PMEPA1 in normal prostate epithelialcells (Clonetics, San Diego, Calif.), prostate cancer cells PC3 (ATCC)and LNCaP cells and breast cancer cells MCF7 (ATCC) was also analyzed bynorthern blot.

[0193] In situ hybridization of PMEPA1 in prostate tissues. A 430 bp PCRfragment (PCR sense primer: 5′CCTTCGCCCAGCGGGAGCGC 3′, (SEQ ID NO. 9)PCR antisense primer 5′CAAGCTCTCTTAGCTTGTGCATTC3′ (SEQ ID NO. 10) wasamplified from cDNA of LNCaP cells treated by R1881 and was cloned intoa PCR-blunt IITOPO vector (Invitrogen, Carlsbad, Calif.). Digoxigeninlabeled antisense and sense riboprobes were synthesized using an invitro RNA transcription kit (Boehringer Mannheim, GMbH, Germany) and alinearized plasmid with PMEPA1 gene fragment as templates. Frozen normaland malignant prostate tissues were fixed in 4% paraformaldehyde for 30min. Prehybridization and hybridization were performed at 55° C. Afterhybridization, slides were sequentially washed with 2×SSC at roomtemperature for 30 min, 2×SSC at 58° C. for 1 hr and 0.1×SSC at 58° C.for 1 hr. Antibody against digoxygenin was used to detect the signal andNBT/BCIP was used as substrate for color development (BoehringerMarnnheim, GMbH, Germany). The slides were evaluated under an OlympusBX-60 microscope. Full-length PMEPA1 coding sequence and chromosomallocalization.

[0194] Analysis of the 1,141 bp PMEPA1 cDNA sequence (SEQ ID NO.1)revealed an open reading frame of 759 bp nucleotides (SEQ ID NO. 2)encoding a 252 amino acid protein (SEQ ID NO. 3) with a predictedmolecular mass of 27.8 kDa, as set forth below in Table 1. TABLE 1 (SEQID NO. 1)TCCTTGGGTTCGGGTGAAAGCGCCTGGGGGTTCGTGGCCATGATCCCCGAGCTGCTGGAGAACTGAAGGCGGACAGTCTCCTGCGAAAC90          ▾AGGCAATGGCGGAGCTGGAGTTTGTTCAGATCATCATCATCGTGGTGGTGATGATGGTGATGGTGGTGGTGATCACGTGCCTGCTGAGCC180 (SEQ ID NO. 3)      M  A  E  L  E  F  V  Q  I  I  I  I  V  V  V  M  M  V  M  V  V  V  I  T  C  L  L  S28                                                                                   ▾ACTACAAGCTGTCTGCACGGTCCTTCATCAGCCGGCACAGCCAGGGGCGGAGGAGAGAAGATGCCCTGTCCTCAGAAGGATGCCTGTGGC270H  Y  K  L  S  A  R  S  F  I  S  R  H  S  Q  G  R  R  R  E  D  A  L  S  S  E  G  C  L  W58                                          ▾CCTCGGAGAGCACAGTGTCAGGCAACGGAATCCCAGAGCCGCAGGTCTACGCCCCGCCTCGGCCCACCGACCGCCTGGCCGTGCCGCCCT360P  S  E  S  T  V  S  G  N  G  I  P  E  P  Q  V  Y  A  P  P  R  P  T  D  R  L  A  V  P  P88TCGCCCAGCGGGAGCGCTTCCACCGCTTCCAGCCCACCTATCCGTACCTGCAGCACGAGATCGACCTGCCACCCACCATCTCGCTGTCAG450F  A  Q  R  E  R  F  H  R  F  Q  P  T  Y  P  Y  L  Q  H  E  I  D  L  P  P  T  I  S  L  S118ACGGGGAGGAGCCCCCACCCTACCAGGGCCCCTGCACCCTCCAGCTTCGGGACCCCGAGCAGCAGCTGGAACTGAACCGGGAGTCGGTGC540D  G  E  E  P  P  P  Y  Q  G  P  C  T  L  Q  L  R  D  P  E  Q  Q  L  E  L  N  R  E  S  V148GCGCACCCCCAAACAGAACCATCTTCGACAGTGACCTGATGGATAGTGCCAGGCTGGGCGGCCCCTGCCCCCCCAGCAGTAACTCGGGCA630R  A  P  P  N  R  T  I  F  D  S  D  L  M  D  S  A  R  L  G  G  P  C  P  P  S  S  N  S  G178TCAGCGCCACGTGCTACGGCAGCGGCGGGCGCATGGAGGGGCCGCCGCCCACCTACAGCGAGGTCATCGGCCACTACCCGGGGTCCTCCT720I  S  A  T  C  Y  G  S  G  G  R  M  E  G  P  P  P  T  Y  S  E  V  I  G  H  Y  P  G  S  S208TCCAGCACCAGCAGAGCAGTGGGCCGCCCTCCTTGCTGGAGGGGACCCGGCTCCACCACACACACATCGCGCCCCTAGAGAGCGCAGCCA810F  Q  H  Q  Q  S  S  G  P  P  S  L  L  E  G  T  R  L  H  H  T  H  I  A  P  L  E  S  A  A238TCTGGAGCAAAGAGAAGGATAAACAGAAAGGACACCCTCTCTAGGGTCCCCAGGGGGGCCGGGCTGGGGCTGCGTAGGTGAAAAGGCAGA900 I  W  S  K  E  K  D  K  Q  K  G  H  P  L  * 252ACACTCCGCGCTTCTTAGAAGAGGAGTGAGAGGAAGGCGGGGGGCGCAGCAACGCATCGTGTGGCCCTCCCCTCCCACCTCCCTGTGTAT990AAATATTTACATGTGATGTCTGGTCTGAATGCACAAGCTAAGAGAGCTTGCAAAAAAAAAAAGAAAAAAGAAAAAAAAAAACCACGTTTC1080                                                      ▾TTTGTTGAGCTGTGTCTTGAAGGCAAAAGAAAAAAAATTTCTACAGTAAAAAAAAAAAAAA   1141

[0195] As indicated above, Table 1 represents the nucleotide andpredicted amino acid sequence of PMEPA1 (GenBank accession No.AF224278). The potential initiation methionine codon and the translationstop codons are indicated in bold. The transmembrane domain isunderlined. The locations of the intron/exon boundaries are shown witharrows, which were determined by comparison of the PMEPA1 cDNA sequenceto the publicly available sequences of human clones RP5-1059L7 and 718J7(GenBank accession No. AL121913 and AL035541).

[0196] A GenBank search revealed a sequence match of PMEPA1 cDNA to twogenomic clones, RP5-1059L7 (accession number AL121913 in theGenBank/htgc database) and 718J7 (accession number AL035541 in theGenBank/nr database). These two clones mapped to Chromosome20q13.2-13.33 and Chromosome 20q13.31-13.33. This information providedevidence that PMEPA1 is located on chromosome 20q13.

[0197] The intron/exon juctions of PMEPA1 gene were determined based onthe comparison of the sequences of PMEPA1 and the two genomic clones. Aprotein motif search using ProfileScan(http://www.ch.embnet.org/cgi-bin/TMPRED) indicated the existence of atype Ib transmembrane domain between amino acid residues 9 to 25 of thePMEPA1 sequence. Another GenBank search further revealed that the PMEPA1showed homology (67% sequence identity and 70% positives at proteinlevel) to a recently described novel cDNA located on chromosome 18(accession number NM_(—)004338) (Yoshikawa et al., 1998), as set forthbelow in Table 2. In addition to the sequence similarity, PMEPA1 alsoshares other features with C18orf1, e.g., similar size of the predictedprotein and similar transmembrane domain as the 1 isoform of C18orf1.TABLE 2 2 AELEFVQIIIIVVVMMVMVVVITCLLSHYKLSARSFISRHSQGRRREDALSSEGCLWPSE61 PMEPA1 (SEQ ID NO: 11)AELEF QIIIIVVV  V VVVITCLL+HYK+S RSFI+R +Q RRRED L  EGCLWPS+ 3AELEFAQIIIIVVVVTVMVVVIVCLLNHYKVSTRSFINRPNQSRRREDGLPQEGCLWPSD 62 C18orf1(SEQ ID NO: 12) 62STVSGNGIPEPQVYAPPRPTDRLAVPPFAQRERFHRFQPTYPYLQHEIDLPPTISLSDGE 121 PMEPA1S     G  E  +   PR  DR   P F QR+RF RFQPTYPY+QHEIDLPPTISLSDGE 63SAAPRLGASE--IMHAPRSRDRFTAPSFIQRDRFSRFQPTYPYVQHEIDLPPTISLSDGE 120 C18orf1122 EPPPYQGPCTLQLRDPEQQLELNRESVRAPPNRTIFDSDLMDSARL-GGPCPPSSNSGIS 180PMEPA1 EPPPYQGPCTLQLRDPEQQ+ELNRESVRAPPNRTIFDSDL+D A   GGPCPPSSNSGIS 121EPPPYQGPCTLQLRDPEQQMELNRESVRAPPNRTIFDSDLIDIAMYSGGPCPPSSNSGIS 180 C18orf1181 ATCYGSGGRMEGPPPTYSEVIGHYPGSSFQHQQSSGPPSLLEGTRLHHTHIAPLESAAIW 240PMEPA1 A+   S GRMEGPPPTYSEV+GH+PG+SF H Q S   +   G+RL        ES  + 181ASTCSSNGRMEGPPPTYSEVMGHHPGASFLHHQRS---NAHRGSRLQFQQ-NNAESTIVP 236 C18orf1241 SKEKDKQKGH  250 PMEPA1  K KD++ G+ 237 IKGKDRKPGN  246 C18orf1

[0198] Analysis of PMEPA1 Expression

[0199] Northern hybridization revealed two transcripts of ˜2.7 kb and 5kb using either PMEPA1 cDNA or oligo probe. The signal intensity ofbands representing these two transcripts was very similar on the X-rayfilms of the northern blots. RT-PCR analysis of RNA from LNCaP cellswith four pairs of primers covering different regions of PMEPA1 proteincoding region revealed expected size of bands from PCR reactions,suggesting that two mRNA species on northern blot have identicalsequences in the protein coding region and may exhibit differences in 5′and/or 3′non-coding regions. However, the exact relationship between thetwo bands remains to be established. Analysis of multiple northern blotscontaining 23 human normal tissues revealed the highest level of PMEPA1expression in prostate tissue. Although other tissues expressed PMEPA1,their relative expression was significantly lower as compared toprostate (FIG. 1). In situ RNA hybridization analysis of PMEPA1expression in prostate tissues revealed abundant expression in theglandular epithelial compartment as compared to the stromal cells.However, both normal and tumor cells in tissue sections of primary tumortissues revealed similar levels of expression.

[0200] Androgen Dependent Expression of PMEPA1

[0201] As discussed above, PMEPA1 was originally identified as a SAGEtag showing the highest fold induction (29-fold) by androgen. Androgendepletion of LNCaP cells resulted in decreased expression of PMEPA1.Androgen supplementation of the LNCaP cell culture media lackingandrogen caused induction of both ˜2.7 and ˜5.0 bp RNA species of PMEPA1in LNCaP cells in a dose and time dependent fashion (FIG. 2A). Basallevel of PMEPA1 expression was detected in normal prostatic epithelialcell cultures and androgen-dependent LNCaP cells cultured in regularmedium. PMEPA1 expression was not detected in AR negative CaP cells, PC3or in the breast cancer cell line, MCF7 (FIG. 2B).

[0202] Evaluation of PMEPA1 expression in androgen sensitive andandrogen refractory tumors of CWR 22 prostate cancer xenograft model

[0203] Previous studies have described increased expression of ARGs inthe “hormone refractory” CWR22R variants of the CWR22 xenograft,suggesting the activation of AR mediated cell signaling in relapsedCWR22 tumors following castration. The androgen sensitive CWR22 tumorexpressed detectable level of PMEPA1 transcripts. However, three of thefour CWR22R tumors exhibited increased PMEPA1 expression (FIG. 8).

EXAMPLE 8

[0204] Structural Features of the PMEPA1 Gene.

[0205] Analysis of a 1,141 base pair PMEPA1 cDNA sequence revealed anopen reading frame of 759 nucleotides (SEQ ID NO:2) that encodes a 252amino acid protein (SEQ ID NO:3). A protein motif search usingProfileScan (http://www.ch.embnet.org/cAibin/TMPRED) indicated theexistence of a type Ib transmembrane domain between amino acid residues9 to 25 of the PMEPA1 sequence. In addition, the motif search revealedtwo PY motifs in the PMEPA1 protein sequence, PPPY (SEQ ID NO:80)(“PY1”) and PPTY (SEQ ID NO:81) (“PY2”). The PY motif is a proline-richpeptide sequence with a consensus PPXY sequence (where X represents anyamino acid) that can bind to proteins with WW domains [Jolliffe et al.,Biochem. J., 351: 557-565, 2000; Harvey et al., Trends Cell Biol., 9:166-169, 1999; Hicke, Cell, 106: 527-530, 2001; Kumar et al., Biochem.Biophys. Res. Commun., 185: 1155-1161, 1992; Kumar et al., Genomics, 40:435-443, 1997; Sudol, Trends Biochem. Sci., 21: 161-163, 1996; Harvey etal., J. Biol. Chem., 277: 9307-9317, 2002; and Brunschwig et al., CancerRes., 63: 1568-1575, 2003].

[0206] A protein sequence homology search revealed that PMEPA1 has an83% sequence identity with a mouse NEDD4 WW binding protein 4 (“N4WBP4,”Accession number AK008976) (4), as shown below in Table 10. In Table 2,the + denotes a conservative substitution, and the PY motifs areunderlined. TABLE 10 Human PMEPA1: 1MAELEFVQXXXXXXXXXXXXXXXTCLLSHYKLSARSFISRHSQGRRREDALSSEGCLWPS 60 SEQ IDNO. 3 + ELEFVQ               TCLLSHYKLSARSFISRHSQ RRR+D LSSEGCLWPS MouseN4WBP4: 18 ITELEFVQIVVIVVVMMVMVVMITCLLSHYKLSARSFISRHSQARRRDDGLSSEGCLWPS77 SEQ ID NO. 68 Human PMEPA1: 61ESTVSGNGIPEPQVYAPPRPTDRLAVPPFAQRERFHRFQPTYPYLQHEIDLPPTISLSDG 120ESTVSG G+PEPQVYAPPRPTDRLAVPPF QR    RFQPTYPYLQHEI LPPTISLSDG MouseN4WBP4: 78 ESTVSG-GMPEPQVYAPPRPTDRLAVPPFIQRS---RFQPTYPYLQHEIALPPTISLSDG133 Human PMEPA1: 121EEPPPYQGPCTLQLRDPEQQLELNRESVRAPPNRTIFDSDLMDSARLGGPCPPSSNSGIS 180EEPPPYQGPCTLQLRDPEQQLELNRESVRAPPNRTIFDSDL+DS  LGGPCPPSSNSGIS MouseN4WBP4: 134 EEPPPYQGPCTLQLRDPEQQLELNRESVRAPPNRTIFDSDLIDSTMLGGPCPPSSNSGIS193 Human PMEPA1: 181ATCYGSGGRMEGPPPTYSEVIGHYPGSSFQHQQSSGPPSLLEGTRLHHTHIAPLESAAIW 240ATCY SGGRMEGPPPTYSEVIGHYPGSSFQHQQS+GP SLLEGTRLHH+HIAPLE Mouse N4WBP4:194 ATCYSSGGRMEGPPPTYSEVIGHYPGSSFQHQQSNGPSSLLEGTRLHHSHIAPLE----- 248Human PMEPA1: 241 SKEKDKQKGHPL  252 +KEK+KQKGHPL Mouse N4WBP4: 249NKEKEKQKGHPL  260

[0207] The WW domains of NEDD4 protein facilitate its binding to thetarget proteins via interaction with the PY motifs of NEDD4 bindingproteins [Jolliffe et al., Biochem. J., 351: 557-565, 2000; Sudol M,Trends Biochem. Sci., 21: 161-163, 1996; Harvey et al., J. Biol. Chem.,277: 9307-9317, 2002; Macias et al., Nature, 382: 646-649, 1996; Chen etal., Proc. Natl. Acad. Sci., USA., 92: 7819-7823, 1995; and Murillas etal., J. Biol. Chem., 277: 2897-2907, 2002]. The PMEPA1 protein sequencecomprises two PY motifs, i.e., PPPY (SEQ ID NO:80) (“PY1”) and PPTY (SEQID NO:81) (“PY2”). PY1 is in the central region of the PMEPA1 proteinand PY2 is close to the carboxyl terminus of the PMEPA1 protein (Table2). Therefore, the high protein sequence identity of PMEPA1 with N4WBP4and the presence of PY motifs indicates that PMEPA1 is the human homologof N4 WBP4 and can bind to the NEDD4 protein and other proteinscontaining a WW domain.

EXAMPLE 9

[0208] PMEPA1-PY Motifs Interact with the WW Domains of NEDD4

[0209] Plasmids. Mammalian expression vectors encoding PMEPA1-V5 andPMEPA1-GFP fusion proteins were generated by PCR amplification of thePMEPA1 open reading frame. For PMEPA1-V5-pcDNA3.1 vector the followingprimers were used:

[0210] 5′-GCTGCTGGAGAACTGAAGGCG-3′ (SEQ ID NO:69) and

[0211] 5′-GTGTCCTTTCTGTTTATCCTTC-3′ (SEQ ID NO:70).

[0212] For PMEPA1-GFP-pEGFP-vector the primers used were:

[0213] 5′-AAGCTTGCTGCTGGAGAACTGAAGG CG-3′ (SEQ ID NO:71) and

[0214] 5′-GAATTCGGTGTCCTTTCTGTTTATC-3′ (SEQ ID NO:72).

[0215] The V5 tag or GFP protein was fused at the carboxyl terminus ofthe PMEPA1 protein. The PCR product for generating PMEPA1-V5 wasinserted into pcDNA3.1-V5-His expression vector (Invitrogen, Carlsbad,Calif.). The PCR product for generating PMEPA1-GFP was digested byHindIII and EcoRI and cloned into the same sites of pEGFP vector(Clontech, Palo Alto, Calif.). PMEPA1-PY motif mutants, in which thetyrosine residue (Y) was replaced with an alanine residue (A), werecreated by using QuikChange Site-Directed Mutagenesis kit (Stratagene,La Jolla, Calif.) and using the PMEPA1-V5-pcDNA3.1 vector as a template.The plasmids of PMEPA1-PY motif mutants are as follows:PMEPA1-PY1m-V5-pcDNA3.1, with the first PY motif mutation (Y126A),PMEPA1-PY2m-V5-pcDNA3.1, with the second PY motif mutation (Y197A), andPMEPA1-PY1m/PY2m-V5-pcDNA3.1, with both the PY motif mutations (Y126Aand Y197A). The sequences of all the inserts in expression vectors wereverified by DNA sequencing.

[0216] A bacterial expression plasmid of human NEDD4 gene(pNEDD4WW-GSTpGEX-2TK) encoding all four WW-domains (Accession numberXM_(—)046129) fused to glutathione S-transferase (GST-WW fusionprotein), was generated by PCR amplification of the coding region of thefour WW-domains using the primers:

[0217] 5′-GCAGGATCCCAACCAGATGCTGCTTGC-3′ (SEQ ID NO:73) and

[0218] 5′-GCAGAATTCTTTTGTAATCCCTGGAGTA-3′(SEQ ID NO:74).

[0219] Normal prostate tissue derived cDNA was used as a PCR templateand the amplified fragment was cloned into the BamHI/EcoRI sites ofpGEX-2TK (Amersham Biotech, Piscataway, N.J.). A mammalian expressionvector (NEDD4-GFP-pEGFP) encoding NEDD4-GFP fusion protein was generatedusing the following primers to generate the NEDD4 gene fragment by PCR.:

[0220] 5′-GCAAAGCTTGTCCGGTTTGCTGGAAGC-3′ (SEQ ID NO:75) and

[0221] 5′-GCAGAATTCCCTTTTTGTTCTTATTGGTGAC-3′ (SEQ ID NO:76).

[0222] PMEPA™ and NEDD4 Protein Binding Assays. The in vitro binding ofPMEPA1 and NEDD4 was assessed by GST pull-down assays. GST-WW fusionprotein was prepared and purified with glutathione-Sepharose beads perAmersham Biotech instructions. [³⁵S]methionine labeled proteinsrepresenting PMEPA1 and its mutants were generated by in vitrotranscription/translation (TNT T7 quick coupledtranscription/translation system, Promega, Madison, Wis.). Briefly, thePMEPA1-V5-pcDNA3.1 or the three mutants (2 μg) were incubated in 40 μlof reticulocyte lysate with 40 μCi of [³⁵S]methionine for 1.5 hrs at 30°C.

[0223] [³⁵S]methionine incorporation into protein was measured andsamples were equalized on the basis of cpm. The GST-WW fusion proteinbound to glutathione-Sepharose beads (5 μg) was incubated with the[³⁵S]methionine labeled lysates (12 μl) in 0.4 ml of phosphate-bufferedsaline (PBS, pH 7.4), 1 mM dithiothreitol, and protease inhibitors. Thenegative control for each [³⁵S]methionine labeled lysate represented areaction mixture with equivalent amount of the lysate incubated withglutathione-Sepharose beads without GST-WW fusion protein. After 16hours of incubation at 4° C., the beads were washed six times with PBS,resuspended in SDS-PAGE sample buffer and run on 12% SDS-PAGE gel undera reducing condition. The gels were dried and autoradiographed.

[0224] Results. The interaction of PMEPA1 and NEDD4 proteins in cellswas evaluated by a co-immunoprecipitation assay. 293 cells (humanembryonal kidney cells) were co-transfected with NEDD4-GFP-pEGFP vectorand one of the PMEPA1-V5 expression vectors encoding either wt PMEPA1-V5or the PY mutants of PMEPA1. Thirty-six hours later the cells werecollected and lysed and the lysates were immunoprecipitated withanti-GFP antibody (Clontech, Palo Alto, Calif.) following themanufacturer's protocol. The immunoprecipitated proteins were subjectedto immunoblotting with an anti-V5 tag antibody (Invitrogen).

[0225] In vitro translated [³⁵S]Methionine-labeled PMEPA1-V5 fusionprotein, with the two intact PY motifs, showed binding to the GST-WWfusion protein (FIG. 6, lane 1). PMEPA1 with PY1 or PY2 mutationsrevealed significantly decreased binding to WW domains (FIG. 6, lane 2and lane 3). Further, PMEPA1-V5 and NEDD4-GFP fusion proteins expressedin 293 cells showed strong association (FIG. 7, lane 1) and the mutantPMEPA1V5 proteins having single mutation of PY1 or PY2 motif or doublemutations of both PY1 and PY2 motifs exhibited significantly reducedbinding to NEDD4 (FIG. 7, lanes 2, 3, and 4). Thus both in vitro andcell culture data reveal that PMEPA1 interacts with NEDD4 and thisinteraction involves the binding of the PMEPA1 PY motifs to WW domains.The PY2 motif mutation appeared to have a greater effect on binding ofPMEPA1 to the NEDD4 WW domain.

[0226] The high protein sequence identity of PMEPA1 with N4WBP4 suggeststhat PMEPA1 is the human homolog of N4 WBP4.

EXAMPLE 10

[0227] PMEPA1 Down Regulates Androgen Receptor and AffectsTranscriptional Targets of the Androgen Receptor

[0228] LNCaP cells were stably transfected with PMEPA1-GFP(PMEPA-GFP-LNCaP) and pEGFP control (pEGFP-LNCaP) expression vectors. Toevaluate the effects of exogenous PMEPA1 expression on androgen receptorin LNCaP transfectants, cells were maintained in androgen-free media for5 days which is known to down regulate endogenous PMEPA1 expression.Androgen receptor expression was evaluated in these cells after 5 daysin the androgen free media (time, 0 hr). Androgen receptor expressionwas also evaluated in cells replenished with 0.11 nM R1881 for differenttime points (12 hours and 24 hours) after androgen withdrawal. Westernblot analysis revealed reduced expression of androgen receptor proteinin PMEPA-GFP-LNCaP cells (FIG. 4A). Decreased androgen receptor proteinlevels in PMEPA1 transfectants correlated with the reduced levels of PSAprotein, a likely consequence of the attenuation of PSA gene expressiondue to relatively low levels of androgen receptor protein. PMEPA1down-regulation of androgen receptor was further supported by results ofrelative increase of PSMA levels whose expression is normally downregulated by androgen receptor. These experiments showed that PMEPA1down regulated androgen receptor, and androgen receptor transcriptionaltargets were affected correspondingly.

[0229] Because PMEPA1 is a NEDD4 binding protein, its effects onandrogen receptor expression may involve the ubiquitin-proteasomepathway. To show that PMEPA1's effect on androgen receptor expressiondoes not result from a general or non-specific effect of theupregulation of a ubiquitin protein ligase in the protein degradationpathway, we evaluated the effects of PMEPA1 on androgen receptor and thep27 protein, which is known to be degraded through a ubiquitin-dependentpathway. We generated a stable PMEPA1GFP-Tet-LNCaP transfectant, inwhich the expression of PMEPA1-GFP fusion protein is regulated bytetracycline (Tet-off system, Clontech). As shown in FIG. 4B, cellscultured in the medium with tetracycline lacked PMEPA1 expression(Tet-off) but overexpressed PMEPA1 when cultured in the medium withouttetracycline. The protein level of androgen receptor decreaseddramatically in PMEPA1-overexpressing cells as compared to the relativeexpression of p27 or tubulin (FIG. 4B). Taken together, these data showthat androgen receptor is a specific target of PMEPA1.

EXAMPLE 11

[0230] Golgi Association of PMEPA1 Protein.

[0231] Our studies also revealed that PMEPA1 is a Golgi-associatedprotein.

[0232] Immunofluorescence Assays. Plasmids were prepared as discussedabove in Example 9. The immunofluorescent assays were performedfollowing the procedure described by Harvey et al., J. Biol. Chem., 277:9307-9317, 2002. Briefly, stable transfectants of LNCaP cells harboringPMEPA1-GFP-pEGFP (LNCaP-PMEPA1-GFP transfectant) were grown oncoverslips for two days, fixed in 2% paraformaldehyde for 15 minutes andpermeabilized in 0.2% Triton X-100 for 2 minutes. Fixed andpermeabilized cells were incubated with anti-GM130 (recognizes acis-Golgi matrix protein) or anti-TGN38 (recognizes a protein localizingto Trans-Golgi Network, TGN) monoclonal antibodies (BD TransductionLaboratory, San Diego, Calif.) at 6.25 μg/ml for 30 minutes at roomtemperature. Cells were then washed to remove excess or non-specificallybound primary antibody followed by incubation with TRITC conjugatedanti-mouse antibody (Sigma, ST. Louis, Mo.) at 1:100 dilution for 30minutes at room temperature. The sections were mounted with fluoromount(Southern Associates, Birmingham, Ala.) and the images were processedwith a Leica fluoromicroscope and Open-Lab software (Improvision,Lexington, Mass.).

[0233] Results. PMEPA1-GFP fusion protein showed peri-nuclearlocalization with a Golgi-like appearance. The images of sub-cellularlocation of GM130, a cis-Golgi protein, showed similar pattern asPMEPA1-GFP fusion protein. Superimposing the images of PMEPA1-GFP fusionprotein and GM130 in LNCaP-PMEPA1-GFP transfectants confirmed thelocalization of PMEPA1-GFP fusion protein on cis-Golgi structure. We didnot observe the co-localization of PMEPA1-GFP and TGN-38, whichlocalizes to TGN.

[0234] The sub-cellular localization of PMEPA1 is similar to two othernewly identified NEDD4 WW domain binding proteins, N4WBP5 and N4WBP5a,which also localize to the Golgi complex [Harvey et al., J. Biol. Chem.,277: 9307-9317, 2002; Konstas et al., J. Biol. Chem., 277: 29406-29416,2002]. N4WBP5a sequestered the trafficking of NEDD4/NEDD4-2 therebyincreasing the activity of the epithelial sodium channel (EnaC), a knowntarget down regulated by NEDD4 [Konstas et al., J. Biol. Chem., 277:29406-29416, 2002]. As a highly androgen-regulated gene and a NEDD4binding protein, the localization of PMEPA1 on the Golgi apparatussuggests that PMEPA1 is involved in protein turn-over of androgenreceptor targets.

EXAMPLE 12

[0235] PMEPA1 Inhibits Growth of Prostate Cancer Cells.

[0236] Colony-Forming Assays. To investigate the biologic effects ofPMEPA1 expression in regulating cell growth and the contribution of PYmotifs to such functions, we performed the colony-formation assay bytransfecting various prostate cancer cell lines with expression vectorsof the wild type PMEPA1 (“wt-PMEPA1”) and PMEPA1-PY mutants.

[0237] Prostate cancer cell lines: LNCaP, PC3, and DU145 were purchasedfrom ATCC (Rockville, Md.) and grown in the cell culture media asdescribed by the supplier. The LNCaP sub-lines C4, C₄₋₂ and C₄₋₂B [Hsiehet al., Cancer Res., 53: 2852-7, 1993; Thalmann et al., Cancer Res., 54:2577-81, 1994; and Wu et al., Int. J. Cancer, 77: 887-94, 1998] werepurchased from Urocor (Oklahoma, Okla.) and cultured in T medium (5%FBS, 80% DMEM, 20% F12, 5 ug/ml insulin, 13.65 pg/ml Triiodo-Thyronine,5 ug/ml apotransferrin, 0.244 ug/ml biotin, 25 ug/ml adenine).

[0238] Three micrograms of plasmids (PMEPA1-V5-pcDNA3.1 or vectorwithout PMEPA1 insert) were transfected into the 50-70% confluent cellsin triplicate in 60-mm petri dishes with Lipofectamine (Invitrogen,Carlsbad, Calif.). Tumor suppressor gene p53 (wt), and mt p53 (R175H andG245D) were also used in parallel as controls. Approximately 36 hourslater, selection with G418 at 800 μg/ml (DU145 and PC3) or 400 μg/ml(LNCaP and its sublines) was initiated. Cells were maintained withG418-containing medium that was changed every 3-4 days. After 2-4 weeksof selection, the cells were rinsed with 1×PBS, fixed with 2%formaldehyde in 1×PBS for 15 minutes, stained with 0.5% crystal violetin 1×PBS for 15 minutes, and rinsed 1-2 times with distilled H₂O.Colonies visible in each dish without magnification were counted byOpen-Lab software.

[0239] To assess the effects of the PY motif mutations on thecolony-forming ability of PMEPA1, LNCaP and PC3 cells were alsotransfected with PMEPA1 mutants: PMEPA1-PY1m-pcDNA3.1,PMEPA1-PY2m-pcDNA3.1, or PMEPA1-PY1m/PY2 m-pcDNA3.1. PMEPA1-V5-pcDNA3.1and expression vector without insert served as positive and negativecontrols, respectively, for the PMEPA1 mutants. Two independentcolony-forming assays were performed as above.

[0240] As shown in FIGS. 3A-F, the colony-forming abilities of prostatecancer cell lines DU145, PC3, LNCaP, and LNCaP sublines weresignificantly suppressed by transfection of the sense version of thewt-PMEPA1 expression vector. Under these conditions wt-p53 showedsimilar cell growth inhibition (data not shown).

[0241] In two independent experiments, mutation of the PY1 motif appearsto abolish the inhibition of colony formation by wt-PMEPA1, emphasizingthe role of the PY1 motif in PMEPA1 and NEDD4 interactions and thebiologic functions of PMEPA1 (FIG. 3G-H). The growth inhibitory effectof PMEPA1 appears to be linked to the interactions of PY1 motif to NEDD4WW domain. This interpretation is based on the striking observationsshowing distinctively more colonies with PY1 motif mutant in comparisonto wt-PMEPA1.

[0242] Cell Proliferation Analysis. To further evaluate the growthinhibitory effects of PMEPA1 on prostate cancer cells, a stablePMEPA1-GFP-Tet LNCaP transfectant was generated. Expression ofPMEPA1-GFP fusion protein in these cells was negatively regulated bytetracycline in the medium (Clontech). For cell proliferation assays,three thousand PMEPA1-GFP-Tet LNCaP cells were seeded in 96-well plateswith or without 1 kg/ml of tetracycline in the medium. CellTiter 96Aqueous One Solution kit (Promega, Madison, Wis.) was used to measurethe cell proliferation according to the manufacturer's instructions.

[0243] The growth inhibitory effect of PMEPA1 has been further confirmedby the cell proliferation characteristics of stable PMEPA1-GFP-Tet-LNCaPcells, where exogenous PMEPA1 is upregulated in the absence oftetracycline. The growth of the PMEPA1-GFP-Tet LNCaP cells intetracycline negative medium is significantly slower than that ofPMEPA1-tet LNCaP transfectant in tetracycline positive medium (FIG. 5).LNCaP cells with PMEPA1 overexpression also revealed increased RBphosphorylation further confirming the cell growth inhibitory effect ofPMEPA1 (data not shown).

[0244] PMEPA1 is expressed in androgen receptor positive prostate cancercell lines, including LNCaP and its sublines (C4, C₄₋₂ and C₄₋₂B). LNCaPcells are androgen dependent for growth. Even though the growth of LNCaPsublines is androgen independent, androgen receptor is critical fortheir proliferation [Zegarra-Moro et al., Cancer Res., 62: 1008-1013,2002]. We observed that overexpression of PMEPA1 by transfecting thePMEPA1 expression vector into LNCaP and its sublines significantlyinhibited the cell proliferation. Since our preliminary observationsshowed that PMEPA1 overexpression in LNCaP cells resulted in alteredexpression of androgen receptor downstream genes (Xu et al. unpublisheddata), we hypothesized that the growth inhibitory effect of PMEPA1 onLNCaP and its sublines may be mediated directly or indirectly throughaffecting androgen receptor functions. Despite the growth inhibitoryeffect on androgen receptor positive prostate cancer cell lines, PMEPA1was also found to inhibit the growth of androgen receptor negativeprostate tumor cells, DU145 and PC3, suggesting that the growthinhibitory effects of PMEPA1 on DU145 and PC3 could be mediated throughalternative mechanisms, e.g., regulation of other nuclear steroidreceptors by PMEPA1. Nonetheless, inhibition of prostate cancer cellgrowth by PMEPA1 implicates PMEPA1 in control of prostate cancerdevelopment.

EXAMPLE 13

[0245] Decreased PMEPA1 Expression in Prostate Tumor Tissues.

[0246] We also evaluated the relationship of alterations in PMEPA1expression to the clinico-pathologic features of prostate cancer.

[0247] Prostate Tissue Specimens, Laser Capture Microdissection (LCM)and Quantitative RT-PCR (QRT-PCR) Assay. Matched prostate cancer andnormal tissues were derived from radical prostatectomy specimens from 62CaP patients treated at Walter Reed Army Medical Center (under anIRB-approved protocol). The procedures of collecting specimens werepreviously described [Xu et al., Cancer Res. 60: 6568-6572, 2000]. Tenmicron frozen sections were prepared and stored at −70° C.Histologically normal prostate epithelial cells and prostate tumor cellsfrom each patient were harvested using LCM equipment according to theprotocol provided by the manufacturer (Arcturus Engineering, MountainView, Calif.).

[0248] Total RNA was prepared from the harvested normal and tumorprostate epithelial cells as previously described [Xu et al., CancerRes. 60: 6568-6572, 2000] and quantified with Fluorometer (Bio-Rad,Hercules, Calif.). QRT-PCR was conducted using 0.1 ng of total RNA frompaired normal and tumor cells. PMEPA1 PCR primers were carefullydesigned that only amplify PMEPA1 but not STAG1, an alternativelyspliced form of PMEPA1 [Rae et al., Mol. Carcinog., 32: 44-53, 2001].The PCR primers were:

[0249] 5′-CATGATCCCCGAGCTGCT-3′ (SEQ ID NO:77) and

[0250] 5′-TGATCTGAACAAACTCCAGCTCC-3′ (SEQ ID NO:78), and the labeledprobe was:

[0251] 5′-AGGCGGACAGTCTCCTGCGAAAC-3′ (SEQ ID NO:79).

[0252] GAPDH gene expression was detected as the internal control (PEApplied Biosystems, Foster, Calif.). Paired triplicate samples (onelacking RT and duplicate with RT) were amplified in 50 μl volumescontaining the manufacturer's recommended universal reagent, properprimers and probe of PMEPA1 or GAPDH using 7700 sequence detectionsystem (PE Applied Biosystems, Foster, Calif.).

[0253] Results were plotted as average cycle threshold (cT) values foreach duplicate sample minus the average duplicate cT values for GAPDH.Differences between matched tumor (T) and normal (N) samples werecalculated using 2exp(cT_(tumor)−cT_(normal)) and expressed as foldchanges in expression. The expression status of PMEPA1 was furthercategorized as either: 1) overexpression in tumor tissue (T>N), definedas 1+(1.5-3 fold), 2+(3.1-10 fold), 3+(10.1-20 fold) and 4+(>20 fold)increased expression as compared with matched normal tissue; 2) reducedexpression in tumor tissue (T<N), defined as 1—(1.5-3 fold), 2—(3.1-10fold), 3—(10.1-20 fold) and 4—(>20 fold) decreased expression ascompared with matched normal tissue; or 3) no change (T=N), defined as 0(<1.5 fold). No detectable PMEPA1 expression in one of the specimens oftumor/normal pairs was scored as 4+for increased or 4—for decreasedexpression.

[0254] Statistical analysis was performed with the SPSS softwarepackage. The association between PMEPA1 expression andclinico-pathological features was analyzed using chi-square tests. TheKaplan-Meier curves were applied to display the PSA-recurrence-freesurvival data. A p value<0.05 was considered as statisticallysignificant.

[0255] The overall expression pattern of PMEPA1 primary prostate canceris shown below in Table 11. TABLE 11 Number of Degree of PMEPA1 PMEPA1Patients/ Expression Expression Group (%) Quantity Number (%) T < N 40(64.5) 1− 11 (27.5) 2− 17 (42.5) 3−  5 (12.5) 4−  7 (17.5) T > N 10(16.1) 1+  6 (60.0) 2+  4 (40.0) 3+ 4+ T = N 12 (19.4) 0  

[0256] Comparison of PMEPA1 expression between tumor and normal cellsrevealed tumor cell associated decreased expression (T<N) in 64.5% tumorspecimens (40 of 62), increased expression (T>N) in 16.1% specimens (10of 62) and no change (T=N) in 19.4% specimens (12 of 62). When theseexpression patterns were stratified by organ-confined (pT2) andnon-organ-confined (pT3) disease, a higher percentage of PMEPA1reduction was seen in pT3 (74%) vs. pT2 (48%). Because the T>N group hasa small number of cases, we combined the T>N group and the T=N group(T>N group). As shown below in Table 12, comparison of theclinico-pathologic parameters between the T<N group and the T>N grouprevealed that the T<N group had a significantly higher percentage ofpatients with pT3 tumors (p=0.035) and more patients in this group had ahigher level of preoperative serum prostate specific antigen (PSA)(p=0.023). TABLE 12 Time to Pathologic PSA Recurrence Stage PSA Range(%) Recurrence after Surgery PMEPA1 (%) ≦4 4.1-10 10.1-20 (%) (month)Expression T2 T3 ng/ml ng/ml ng/ml No Yes Mean ± SE T < N 11 29 1 30 929 11 8.2 ± (27.5) (72.5) (2.5)  (75.0) (22.5) (72.5) (27.5) 3.4 T ≧ N12 10 5 15 2 19 3 18.4 ± (54.5) (45.5) (22.7) (68.2) (9.1)  (86.4)(13.6) 6.3 pValue 0.035 0.023 0.211 0.18

[0257] Out of 62 patients whose tumors were analyzed for PMEPA1expression, 14 patients showed prostate cancer recurrence as defined byserum PSA level equal or higher than 0.2 ng/ml after prostatectomy. Ofthe 14 patients, 11 showed reduced tumor associated PMEPA1 expression(78.5%). Reduced PMEPA1 expression seems to associate with a higherrecurrence rate and a shorter duration to recurrence after surgery, eventhrough the statistical analysis did not reveal a significantdifference. The absence of a significant difference might be due to thesmall number of patients.

[0258] The specification is most thoroughly understood in light of theteachings of the references cited within the specification which arehereby incorporated by reference. The embodiments within thespecification provide an illustration of embodiments of the inventionand should not be construed to limit the scope of the invention. Theskilled artisan readily recognizes that many other embodiments areencompassed by the invention. TABLE 3 Genes Regulated by Androgen: SAGEData Derived from CPDR SAGE Library Accession Description Effect ofAndrogen AA310984 EST Up-regulated by Androgen M26663

prostate-specific antigen mRNA, Up-regulated by Androgen complete cds.*AA508573 Human nucleolin gene, complete cds Up-regulated by AndrogenAB020637 Homo sapiens mRNA for KIAA0830 protein, partial Up-regulated byAndrogen cds. AA280663 EST Up-regulated by Androgen U31657KRAB-associated protein 1 Up-regulated by Androgen AI879709 ESTUp-regulated by Androgen AA602190 EST Up-regulated by Androgen AF035587Homo sapiens X-ray repair cross-complementing Up-regulated by Androgenprotein 2 (XRCC2) AF151898 Homo sapiens CGI-140 protein mRNAUp-regulated by Androgen AA418786 No reliable matches, only see in twolinberary (1 Up-regulated by Androgen each) AI308812 EST Up-regulated byAndrogen X59408 Membrane cofactor protein (CD46, trophoblast-Up-regulated by Androgen lymphocyte cross-reactive antigen) X81817Accessory proteins BAP31/BAP29 Up-regulated by Androgen AF071538

Ets transcription factor PDEF Up-regulated by Androgen (PDEF) mRNA,complete NM_003201 Transcription factor 6-like 1 (mitochondrialUp-regulated by Androgen transcription factor 1-like) U41387 Human Guprotein mRNA, partial cds. Up-regulated by Androgen U58855 Guanylatecyclase 1, soluble, alpha 3 Up-regulated by Androgen X12794 Human v-erbArelated ear-2 gene. Up-regulated by Androgen U88542

homeobox protein Nkx3.1 Up-regulated by Androgen D89729 Homo sapiensmRNA for CRM1 protein, complete Up-regulated by Androgen cds. U75329TMPRSS2 Up-regulated by Androgen AA062976 EST Up-regulated by AndrogenL12168 Homo sapiens adenylyl cyclase-associated protein Up-regulated byAndrogen (CAP) mRNA AA043945 EST Up-regulated by Androgen AF026291 Homosapiens chaperonin containing t-complex Up-regulated by Androgenpolypeptide 1, delta AB002301 Human mRNA for KIAA0303 gene, partial cds.Up-regulated by Androgen D13643 Human mRNA for KIAA0018 gene, completecds. Up-regulated by Androgen AI310341 EST Up-regulated by AndrogenU49436 Human translation initiation factor 5 (eIF5) mRNA, Up-regulatedby Androgen complete cds S79862 Proteasome (prosome, macropain) 26Ssubunit, non- Up-regulated by Androgen ATPase, 5 M14200 Human diazepambinding inhibitor (DBI) mRNA, Up-regulated by Androgen complete cds.AA653318 FK506-binding protein 5 Up-regulated by Androgen L07493 Homosapiens replication protein A 14 kDa subunit Up-regulated by Androgen(RPA) mRNA, AJ011916 Homo sapiens mRNA for hypothetical protein.Up-regulated by Androgen AA130537 EST Up-regulated by Androgen D16373Human mRNA for dihydrolipoamide Up-regulated by Androgensuccinyltransferase, complete cds. AL096857 Novel human mRNA fromchromosome 1 Up-regulated by Androgen AF007157 Homo sapiens clone 23856unknown mRNA, partial Up-regulated by Androgen cds. AA425929 NADHdehydrogenase (ubiquinone) 1 beta Up-regulated by Androgen subcomplex,10 (22 kD, PDSW) AI357815 EST Up-regulated by Androgen D83778 Human mRNAfor KIAA0194 gene, partial cds. Up-regulated by Androgen AF000979 Homosapiens testis-specific Basic Protein Y 1 Up-regulated by Androgen(BPY1) mRNA, AA889510 EST Up-regulated by Androgen AB018330 Homo sapiensmRNA for KIAA0787 protein, partial Up-regulated by Androgen cds.AA026941 EST Up-regulated by Androgen AA532377 Chromosome 1 open readingframe 8 Up-regulated by Androgen AF010313 Homo sapiens Pig8 (PIG8) mRNA(etoposide- Up-regulated by Androgen induced mRNA), complete cds. L06328Human voltage-dependent anion channel isoform 2 Up-regulated by Androgen(VDAC) mRNA, U41804 Human putative T1/ST2 receptor binding proteinUp-regulated by Androgen precursor mRNA, AB020676 Homo sapiens mRNA forKIAA0869 protein, partial Up-regulated by Androgen cds. J03503 Humanpyruvate dehydrogenase E1-alpha subunit Up-regulated by Androgen mRNA,cds. AA421098 EST Up-regulated by Androgen AF072836 Sox-liketranscriptional factor Up-regulated by Androgen AA115355 ESTUp-regulated by Androgen AF118240 Homo sapiens, peroxisomal biogenesisfactor 16 Up-regulated by Androgen (PEX16) mRNA, complete AA011178 ESTUp-regulated by Androgen X15573 Human liver-type 1-phosphofructokinase(PFKL) Up-regulated by Androgen mRNA, complete cds. AA120930 ESTUp-regulated by Androgen AB002321 Human mRNA for KIAA0323 gene, partialcds Up-regulated by Androgen AF151837 Homo sapiens CGI-79 protein mRNA,complete cds Up-regulated by Androgen AA481027 EST Up-regulated byAndrogen AA039343 EST Up-regulated by Androgen U09716 Humanmannose-specific lectin (MR60) mRNA, Up-regulated by Androgen completecds. AF044773 Homo sapiens breakpoint cluster region protein 1Up-regulated by Androgen (BCRG1) mRNA U51586 Human siah binding protein1 (SiahBP1) mRNA, Up-regulated by Androgen partial cds. M36341 HumanADP-ribosylation factor 4 (ARF4) mRNA, Up-regulated by Androgen completecds. AI282096 EST Up-regulated by Androgen W45510 RAB7, member RASoncogene family-like 1 Up-regulated by Androgen X16135 Human mRNA fornovel heterogeneous nuclear RNP Up-regulated by Androgen protein, Lprotein AF052134 Homo sapiens clone 23585 mRNA sequence, Up-regulated byAndrogen AF052134 D26068 Williams-Beuren syndrome chromosome region 1Up-regulated by Androgen X69433 H. sapiens mRNA for mitochondrialisocitrate Up-regulated by Androgen dehydrogenase (NADP+). X61123 B-celltranslocation gene 1, anti-proliferative Up-regulated by Androgen X63423H. sapiens mRNA for delta-subunit of mitochondrial Up-regulated byAndrogen F1F0 ATP-synthase AJ010025 Homo sapiens mRNA forunr-interacting protein. Down-regulated by Androgen AF003938 Homosapiens thioredoxin-like protein mRNA, Down-regulated by Androgencomplete cds. AB014536 Homo sapiens copine III (CPNE3) mRNADown-regulated by Androgen AA504468 EST Down-regulated by AndrogenNM_001273 Chromodomain helicase DNA binding protein 4 Down-regulated byAndrogen AA015746 Homo sapiens mRNA; cDNA DKFZp586H0722 Down-regulatedby Androgen (from clone DKFZp586H0722) AA552354 EST Down-regulated byAndrogen AA025744 3-prime-phosphoadenosine 5-prime-phosphosulfateDown-regulated by Androgen synthase 2 X71129 H. sapiens mRNA forelectron transfer flavoprotein Down-regulated by Androgen beta subunitAA046050 EST Down-regulated by Androgen U57052 Human Hoxb-13 mRNA,complete cds Down-regulated by Androgen AA400137 EST Down-regulated byAndrogen AA487586 EST Down-regulated by Androgen J04208 Humaninosine-5′-monophosphate dehydrogenase Down-regulated by Androgen (IMP)mRNA M64722 Testosterone-repressed prostate message 2 Down-regulated byAndrogen (apolipoprotein J) AI743483 EST Down-regulated by AndrogenAA476914 EST Down-regulated by Androgen AA026691 EST Down-regulated byAndrogen AI014986 EST Down-regulated by Androgen X85373 Small nuclearribonucleoprotein polypeptide G Down-regulated by Androgen U07231 G-richRNA sequence binding factor 1 Down-regulated by Androgen T97753 Glycogensynthase 2 (liver) Down-regulated by Androgen AA234050 ESTDown-regulated by Androgen AI015143 EST Down-regulated by AndrogenU09196 Human 1.1 kb mRNA upregulated in retinoic acid Down-regulated byAndrogen treated HL-60 neutrophilic cells. AA977749 EST Down-regulatedby Androgen NM_006451 Polyadenylate binding protein-interacting protein1 Down-regulated by Androgen AI818296 EST Down-regulated by AndrogenAI250561 EST Down-regulated by Androgen AA063613 EST Down-regulated byAndrogen U59209 Hs.183596: UDP glycosyltransferase 2 family,Down-regulated by Androgen polypeptide B17, U59209 Z11559Iron-responsive element binding protein 1 Down-regulated by AndrogenAF052578 Homo sapiens androgen receptor associated proteinDown-regulated by Androgen 24 (ARA24) X16312 Human mRNA forphosvitin/casein kinase II beta Down-regulated by Androgen subunit.H17890 PCTAIRE protein kinase 3 Down-regulated by Androgen AA192312 ESTDown-regulated by Androgen AA043787 EST Down-regulated by AndrogenAI052020 EST Down-regulated by Androgen AB014512 Homo sapiens mRNA forKIAA0612 protein Down-regulated by Androgen NM_001328 Homo sapiensC-terminal binding protein 1 (CTBP1) Down-regulated by Androgen mRNAM15919 Human autoimmune antigen small nuclear Down-regulated by Androgenribonucleoprotein E mRNA. AF151813 Homo sapiens CGI-55 protein mRNA,complete cds Down-regulated by Androgen L41351 Protease, serine, 8(prostasin) Down-regulated by Androgen AF077046 Homo sapiens gangliosideexpression factor 2 (GEF- Down-regulated by Androgen 2) homolog U15008Small nuclear ribonucleoprotein D2 polypeptide Down-regulated byAndrogen (16.5 kD), AA938995 N62491 Folate hydrolase (prostate-specificmembrane Down-regulated by Androgen antigen) 1 AI569591 ESTDown-regulated by Androgen AJ131245 Secretory protein 24 (SEC24).Down-regulated by Androgen U90543 Human butyrophilin (BTF1) mRNA,complete cds. Down-regulated by Androgen Z47087 Transcription elongationfactor B (SIII), polypeptide Down-regulated by Androgen 1-like M34539FK506-binding protein 1A (12 kD) Down-regulated by Androgen N43807yy19a05.r1 Soares melanocyte 2NbHM Homo Down-regulated by Androgensapiens cDNA clone U03269 Human actin capping protein alpha subunit(CapZ) Down-regulated by Androgen mRNA, complete AI571685 ESTDown-regulated by Androgen AA010412 EST Down-regulated by AndrogenL40403 Homo sapiens (clone zap3) mRNA, 3′ end of cds. Down-regulated byAndrogen NM_006560 CUG triplet repeat, RNA-binding protein 1Down-regulated by Androgen NM_004713 Serologically defined colon cancerantigen 1 Down-regulated by Androgen U36188Clathrin-associated/assembly/adaptor protein, Down-regulated by Androgenmedium 1 AB020721 KIAA0914 gene product Down-regulated by AndrogenT35365 EST Down-regulated by Androgen AF029789 Homo sapiensGTPase-activating protein (SIPA1) Down-regulated by Androgen mRNA,complete cds. AA427857 EST Down-regulated by Androgen AA910404 ESTDown-regulated by Androgen L42379 Quiescin Q6 (bone-derived growthfactor) Down-regulated by Androgen AL117641 cDNA DKFZp434L235Down-regulated by Androgen AI688119 EST Down-regulated by AndrogenAA688073 EST Down-regulated by Androgen NM_002945 Replication protein A1(70 kD) Down-regulated by Androgen AI797610 EST Down-regulated byAndrogen AF086095 Homo sapiens full length insert cDNA cloneDown-regulated by Androgen YZ88A07. AF070666 Homo sapiens tissue-typepituitary Kruppel- Down-regulated by Androgen associated box proteinR55128 Proteasome (prosome, macropain) 26 S subunit, non- Down-regulatedby Androgen ATPase, 2 X75621 Tuberous sclerosis 2 Down-regulated byAndrogen AA019070 EST Down-regulated by Androgen AI089867 ESTDown-regulated by Androgen NM_001003 Homo sapiens ribosomal protein,large, P1 (RPLP1) Down-regulated by Androgen mRNA L05093 Ribosomalprotein L18a Down-regulated by Androgen AA854176 EST Down-regulated byAndrogen AI929622 Homo sapiens clone 23675 mRNA sequence Down-regulatedby Androgen AI264769 ESTs, Weakly similar to ORF YDL087c Down-regulatedby Androgen [S. cerevisiae] L09159 Ras homolog gene family, member A,may be Down-regulated by Androgen androgen regulated? AI143187 ESTDown-regulated by Androgen H17900 cDNA DKFZp586H051 (from cloneDown-regulated by Androgen DKFZp586H051) NM_005617 Ribosomal protein S14Down-regulated by Androgen L49506 Cyclin G2 Down-regulated by AndrogenAA614448 Regulator of G-protein signalling 5 Down-regulated by AndrogenS83390 T3 receptor-associating cofactor-1 Down-regulated by AndrogenAA917672 EST Down-regulated by Androgen X52151 Arylsulphatase ADown-regulated by Androgen U09646 Carnitine palmitoyltransferase IIDown-regulated by Androgen Z50853 ATP-dependent protease ClpAP (E.coli), proteolytic Down-regulated by Androgen subunit, human AB023208MLL septin-like fusion Down-regulated by Androgen U92014 Human clone121711 defective mariner transposon Down-regulated by Androgen Hsmar2mRNA AA878293 Alpha-1-antichymotrypsin Down-regulated by AndrogenAA554191 EST Down-regulated by Androgen M55618 Hexabrachion (tenascin C,cytotactin) Down-regulated by Androgen AA027050 EST Down-regulated byAndrogen AF112472 Homo sapiens calcium/calmodulin-dependent proteinDown-regulated by Androgen kinase II beta AA583866 EST Down-regulated byAndrogen AA115687 EST Down-regulated by Androgen AA043318 ESTDown-regulated by Androgen U90329 Poly(rC)-binding protein 2Down-regulated by Androgen Y00815 Protein tyrosine phosphatase, receptortype, F Down-regulated by Androgen X76013 H. sapiens QRSHs mRNA forglutaminyl-tRNA Down-regulated by Androgen synthetase. X75861 Testisenhanced gene transcript Down-regulated by Androgen AA593078 Homosapiens PAC clone DJ0167F23 from 7p15 Down-regulated by Androgen J04058Human electron transfer flavoprotein alpha-subunit Down-regulated byAndrogen mRNA AF026292 Homo sapiens chaperonin containing t-complexDown-regulated by Androgen polypeptide 1, eta AF068754 Homo sapiens heatshock factor binding protein 1 Down-regulated by Androgen HSBP1 mRNA,NM_000172 Guanine nucleotide binding protein (G protein), Down-regulatedby Androgen alpha transducing activity polypeptide 1 AI140631 Hs.1915:folate hydrolase (prostate-specific Down-regulated by Androgen membraneantigen) 1

[0259] TABLE 4 Potential Prostate Specific/Abundant Genes Derived FromNCBI and CPDR SAGE Libraries Accession Description M88700 Human dopadecarboxylase (DDC) gene, complete cds. W45526 zc26b04.r1Soares_senescent_fibroblasts_NbHSF Homo sapiens cDNA, Hs.108981: ficolin(collagen/fibrinogen domain-containing) 1, AF201077 NADH: ubiquinoneoxidoreductase MLRQ subunit (NDUFA4) mRNA, complete cds with polyA.D55953 HUM407H12B Clontech human fetal brain polyA + mRNA (#6535) Homo,Hs.118724: histidine triad nucleotide-binding protein, AJ012499, mRNAactivated in tumor suppression, clone TSAP19 with polyA AA082804zn41g02.r1 Stratagene endothelial cell 937223 Homo sapiens cDNA,Hs.110967: ESTs, Weakly similar to KIAA0762 protein [H. sapiens],Hs.5662: guanine nucleotide binding protein (G protein), betapolypeptide 2-like 1 in the sequence no tag X05332 Human mRNA forprostate specific antigen.* AI278854 qo42f01.x1 NCI_CGAP_Lu5 Homosapiens cDNA clone IMAGE: 1911193 3′, NM_004537, nucleosome assemblyprotein 1-like 1 (NAP1L1), tag is at beginning of the gene. W75950zd58b02.r1 Soares_fetal_heart_NbHH19W Homo sapiens cDNA clone, AF151840,CGI- 82 protein mRNA, tag is at 3′ end. F02980 HSC1IC062 normalizedinfant brain cDNA Homo sapiens cDNA clone M99487 Human prostate-specificmembrane antigen (PSM) mRNA, complete cds. AL035304 H. sapiens gene fromPAC 295C6, similar to rat PO44. AI088979 ou86f03.s1Soares_NSF_F8_9W_OT_PA_P_S1 Homo sapiens cDNA clone AF186249

six transmembrane epithelial antigen of prostate (STEAP1) mRNA C15801C15801 Clontech human aorta polyA + mRNA (#6572) Homo sapiens cDNAL10340 Human elongation factor-1 alpha (ef-1) mRNA, 3′ end. NM_004540Homo sapiens neural cell adhesion molecule 2 (NCAM2) AA151796 zl39c02.r1Soares_pregnant_uterus_NbHPU Homo sapiens cDNA clone NM_001634 Homosapiens S-adenosylmethionine decarboxylase 1 (AMD1) NM_005013 Homosapiens nucleobindin 2 (NUCB2)AL121913 in GenBank htgc database) and718J7 (Accession number AL035541 AF004828 Homo sapiens rab3-GAPregulatory domain mRNA, complete cds. X60819 X60 H. sapiens DNA formonoamine oxidase type A (14) (partial). AA133972 zl38g12.r1Soares_pregnant_uterus_NbHPU Homo sapiens cDNA clone M69226 Humanmonoamine oxidase (MAOA) mRNA, complete cds. AA969141 op50c11.s1Soares_NFL_T_GBC_S1 Homo sapiens cDNA clone AA523652 ni64d09.s1NCI_CGAP_Pr12 Homo sapiens cDNA clone IMAGE: 981617, mRNA AF078749 Homosapiens organic cation transporter 3 (SLC22A3) AA583544 nf25h10.s1NCI_CGAP_Pr1 Homo sapiens cDNA clone IMAGE: 914851, mRNA AF051894 Homosapiens 15 kDa selenoprotein mRNA, complete cds. AF165967 Homo sapiensDDP-like protein mRNA X57129 H. sapiens H1.2 gene for histone H1.AA640928 nr28d08.r1 NCI_CGAP_Pr3 Homo sapiens cDNA clone IMAGE: 1169295,mRNA U41766 Human metalloprotease/disintegrin/cysteine-rich proteinprecursor AF023676 Homo sapiens lamin B receptor homolog TM7SF2 (TM7SF2)mRNA, U10691 Human MAGE-6 antigen (MAGE6) gene, complete cds. M22976Human cytochrome b5 mRNA, 3′ end. L14778 Human calmodulin-dependentprotein phosphatase catalytic subunit AF071538

Ets transcription factor PDEF (PDEF) mRNA, complete U39840 Humanhepatocyte nuclear factor-3 alpha (HNF-3 alpha) mRNA, AA532511nj54d03.s1 NCI_CGAP_Pr9 Homo sapiens cDNA clone IMAGE: 996293, mRNAX07166 Human mRNA for enkephalinase (EC 3.4.24.11). M96684 H. sapiensPur (pur-alpha) mRNA, complete cds. AI204040 qe77f05.x1Soares_fetal_lung_NbHL19W Homo sapiens cDNA clone AA577923 nl20a01.s1NCI_CGAP_HSC1 Homo sapiens cDNA clone IMAGE: 1041192, AA569633nm38h09.s1 NCI_CGAP_Pr4.1 Homo sapiens cDNA clone IMAGE: 1062497, U65011Human preferentially expressed antigen of melanoma (PRAME) mRNA, U21910Human basic transcription factor BTF2p44 mRNA, 3′ end, partial cds.AA633187 nq07c12.s1 NCI_CGAP_Lu1 Homo sapiens cDNA clone IMAGE: 11431903′ AF000993 Homo sapiens ubiquitous TPR motif, X isoform (UTX) mRNA,W76105 zd65b04.r1 Soares_fetal_heart_NbHH19W Homo sapiens cDNA cloneH39906 yo54a07.r1 Soares breast 3NbHBst Homo sapiens cDNA clone AA971717op95c11.s1 NCI_CGAP_Lu5 Homo sapiens cDNA clone IMAGE: 1584596 3′,M68891 Human GATA-binding protein (GATA2) mRNA, complete cds. AA310157EST181013 Jurkat T-cells V Homo sapiens cDNA 5′ end, mRNA sequence.X00948 Human mRNA for prepro-relaxin H2. AB018330 Homo sapiens mRNA forKIAA0787 protein, partial cds. AA890637 ak11e11.s1Soares_parathyroid_tumor_NbHPA Homo sapiens cDNA clone M64929 J05 Humanprotein phosphatase 2A alpha subunit mRNA, complete cds. W24341zb81h12.r1 Soares_senescent_fibroblasts_NbHSF Homo sapiens cDNA AA974479od58b03.s1 NCI_CGAP_GCB1 Homo sapiens cDNA clone IMAGE: 1372109 3′R31644 yh69e05.r1 Soares placenta Nb2HP Homo sapiens cDNA clone AA573246nm52c02.s1 NCI_CGAP_Br2 Homo sapiens cDNA clone IMAGE: 1071842 3′,AA507635 ng84b02.s1 NCI_CGAP_Pr6 Homo sapiens cDNA clone IMAGE: 941451,mRNA gb|AF008915 Homo sapiens EVI5 homolog mRNA AL049987 Homo sapiensmRNA; cDNA DKFZp564F112 (from clone DKFZp564F112). U81599

homeodomain protein HOXB13 mRNA AA641596 nr20f05.s1 NCI_CGAP_Pr2 Homosapiens cDNA clone IMAGE: 1168545, mRNA D84295 Human mRNA for possibleprotein TPRDII R13859 yf65d08.r1 Soares infant brain 1NIB Homo sapienscDNA clone M34840 Human prostatic acid phosphatase mRNA, complete cds.AA572913 nm42f12.s1 NCI_CGAP_Pr4.1 Homo sapiens cDNA clone IMAGE:1062863, AA094460 cp0378.seq.F Human fetal heart, Lambda ZAP ExpressHomo sapiens AF031166 Homo sapiens SRp46 splicing factor retropseudogenemRNA. AA625147 af70c07.r1 Soares_NhHMPu_S1 Homo sapiens cDNA cloneIMAGE: 1047372 T39510 ya06h07.r1 Stratagene placenta (#937225) Homosapiens cDNA clone R35034 yg60h03.r1 Soares infant brain 1NIB Homosapiens cDNA clone AI003674 zg01c04.s1 Soares_pineal_gland_N3HPG Homosapiens cDNA clone AJ003636 AJ003636 Selected chromosome 21 cDNA libraryHomo sapiens cDNA AA601385 no16d12.s1 NCI_CGAP_Phe1 Homo sapiens cDNAclone IMAGE: 1100855 3′, AF191339 Homo sapiens anaphase-promotingcomplex subunit 5 (APC5) AA431822 zw79d02.r1 Soares_testis_NHT Homosapiens cDNA clone IMAGE: 782403 NM_003909 Homo sapiens copine III(CPNE3) AA484004 ne73f04.s1 NCI_CGAP_Ew1 Homo sapiens cDNA clone IMAGE:909919 AA535774 nj78f08.s1 NCI_CGAP_Pr10 Homo sapiens cDNA clone IMAGE:998631, mRNA NM_000944.1 Homo sapiens protein phosphatase 3 (formerly2B) AA702811 zi90c11.s1 Soares_fetal_liver_spleen_1NFLS_S1 Homo sapienscDNA X95073 H. sapiens mRNA for translin associated protein X. AA029039zk12b07.s1 Soares_pregnant_uterus_NbHPU Homo sapiens cDNA clone AF032887Homo sapiens forkhead (FKHRL1P1) pseudogene N46609 yy48h08.r1Soares_multiple_sclerosis_2NbHMSP Homo sapiens cDNA U58855 Homo sapienssoluble guanylate cyclase large subunit (GC-S-alpha-1) AA255486zr83d03.s1 Soares_NhHMPu_S1 Homo sapiens cDNA clone IMAGE: 682277AA128153 zl15c06.s1 Soares_pregnant_uterus_NbHPU Homo sapiens cDNA cloneAA016039 ze31c05.s1 Soares retina N2b4HR Homo sapiens cDNA clone R88520ym91e09.s1 Soares adult brain N2b4HB55Y Homo sapiens cDNA clone M26624Human CALLA/NEP gene encoding neutral endopeptidase, exon 20. AA026997ze99e01.r1 Soares_fetal_heart_NbHH19W Homo sapiens cDNA clone W48775zc44b08.r1 Soares_senescent_fibroblasts_NbHSF Homo sapiens cDNA AA074407zm15c08.r1 Stratagene pancreas (#937208) Homo sapiens cDNA clone L13972Homo sapiens beta-galactoside alpha-2,3-sialyltransferase (SIAT4A)D14661 Human mRNA for KIAA0105 gene, complete cds. AA115452 zk89a08.r1Soares_pregnant_uterus_NbHPU Homo sapiens cDNA clone AA495742 zw04b12.r1Soares_NhHMPu_S1 Homo sapiens cDNA clone IMAGE: 768287 5′ R13416yf75h09.r1 Soares infant brain 1NIB Homo sapiens cDNA clone AA046369zk77h07.r1 Soares_pregnant_uterus_NbHPU Homo sapiens cDNA clone T35440EST85129 Human Lung Homo sapiens cDNA 5′ end similar to None, mRNAAI075860 oz25b05.x1 Soares_total_fetus_Nb2HF8_9w Homo sapiens cDNA cloneW56437 zc57g05.r1 Soares_parathyroid_tumor_NbHPA Homo sapiens cDNA cloneAI583880 tt70b02.x1 NCI_CGAP_HSC3 Homo sapiens cDNA clone IMAGE: 22460913′, D85181 Homo sapiens mRNA for fungal sterol-C5-desaturase homolog,complete AF105714 Homo sapiens protein kinase PITSLRE (CDC2L2) gene,exon 3. AA401802 zt60c12.r1 Soares_testis_NHT Homo sapiens cDNA cloneIMAGE: 726742 AB002301 Human mRNA for KIAA0303 gene, partial cds. U75667Human arginase II mRNA, complete cds. AA585183 JTH089 HTCDL1 Homosapiens cDNA 5′/3′, mRNA sequence. AF191771 Homo sapiens CED-6 protein(CED-6) mRNA AA650252 ns93g05.s1 NCI_CGAP_Pr3 Homo sapiens cDNA cloneIMAGE: 1191224, mRNA R64618 yi19b09.r1 Soares placenta Nb2HP Homosapiens cDNA clone N24627 yx89a09.s1 Soares melanocyte 2NbHM Homosapiens cDNA clone AB028951 Homo sapiens mRNA for KIAA1028 proteinN75608 yw37a07.r1 Morton Fetal Cochlea Homo sapiens cDNA clone N53899yy98e03.r1 Soares_multiple_sclerosis_2NbHMSP Homo sapiens cDNA N46696yy50f07.r1 Soares_multiple_sclerosis_2NbHMSP Homo sapiens cDNA AA419522zv03d05.r1 Soares_NhHMPu_S1 Homo sapiens cDNA clone IMAGE: 752553 M61906Human P13-kinase associated p85 mRNA sequence. C16570 C16570 Clontechhuman aorta polyA + mRNA (#6572) Homo sapiens cDNA X63105 H. sapiens tprmRNA. AA315855 EST187656 Colon carcinoma (HCC) cell line II Homo sapienscDNA 5′ L18920 Human MAGE-2 gene exons 1-4, complete cds. M25161 HumanNa, K-ATPase beta subunit (ATP1B) gene AA164865 zq41g07.r1 StratagenehNT neuron (#937233) Homo sapiens cDNA clone N40094 yx98g07.r1 Soaresmelanocyte 2NbHM Homo sapiens cDNA clone N98940 yy71a07.r1Soares_multiple_sclerosis_2NbHMSP Homo sapiens cDNA AF049907 Homosapiens zinc finger transcription factor (ZNF-X) mRNA, M78806 EST00954Hippocampus, Stratagene (cat. #936205) Homo sapiens cDNA AA040819zk47b03.r1 Soares_pregnant_uterus_NbHPU Homo sapiens cDNA clone C15445C15445 Clontech human aorta polyA + mRNA (#6572) Homo sapiens cDNAAB018309 Homo sapiens mRNA for KIAA0766 protein, complete cds. AJ011497Homo sapiens mRNA for Claudin-7. X00949 Human mRNA for prepro-relaxinH1. AA418633 zv93d09.r1 Soares_NhHMPu_S1 Homo sapiens cDNA clone IMAGE:767345 5′ AI146806 qb83h04.x1 Soares_fetal_heart_NbHH19W Homo sapienscDNA clone X82942 H. sapiens satellite 3 DNA. AA456383 aa14f03.r1Soares_NhHMPu_S1 Homo sapiens cDNA clone IMAGE: 813245 AA019341ze57e04.s1 Soares retina N2b4HR Homo sapiens cDNA clone AB027466 Homosapiens SPON2 mRNA for spondin 2 AF038170 Homo sapiens clone 23817 mRNAsequence. NM_000240 Homo sapiens monoamine oxidase A (MAOA) N34126yx76c01.r1 Soares melanocyte 2NbHM Homo sapiens cDNA clone N41339yw68g06.r1 Soares_placenta_8to9weeks_2NbHP8to9W Homo sapiens cDNA R34783yh87b05.r1 Soares placenta Nb2HP Homo sapiens cDNA clone N75858yw32a03.r1 Morton Fetal Cochlea Homo sapiens cDNA clone AA633887ac32h04.s1 Stratagene hNT neuron (#937233) Homo sapiens cDNA cloneN53723 yz06d03.r1 Soares_multiple_sclerosis_2NbHMSP Homo sapiens cDNAAI187365 qf29b12.x1 Soares_testis_NHT Homo sapiens cDNA clone IMAGE:1751423

[0260] TABLE 5 Genes/ESTs as Defined by Publications: Including AndrogenSignaling, Prostate Specificity, Prostate Cancer Association, andNuclear Receptors/Regulators with Potential Interaction with AndrogenReceptor Cluster ID Gene Name Description References Hs.81988 DOC-2deliion of ovarian Up-regulated by Androgen Ablation Endocrinology,carcinoma 2 139,3542,98 Hs.155389 RAR a Up-regulated by AndrogenAblation endocrinology, 138,553,97 Hs.12601 AS3 DNA binding proteinUp-regulated by Androgen Ablation J Steroid Biochem Mol Biol 68,41,99Hs.181426 EST Up-regulated by Androgen Ablation Hs.2391 apical proteinUp-regulated by Androgen Ablation Hs.109530 KGF/FGF7 keratinocyte growthfactor Up-regulated by Androgen BBRC 220,858,96, Can Res, 54,5474,94Hs.1104 TGF beta 1 Up-regulated by Androgen Endocrinology, 137,99,96,Endocrinology, 139,378,98 Hs.75525 Calreticulin CalreticulinUp-regulated by Androgen Can Res 59,1896,99 Hs.78888 DBI/ACBPDiazepam-binding Up-regulated by Androgen JBC, 237,19938,98inhibitor/acyl-CoA binding Protein Hs.41569 Phosphatidic acidUp-regulated by Androgen JBC, 273,4660,98 phosphatase type 2a isozymeHs.83190 Fatty acid synthase Up-regulated by Androgen Can Res,57,1086,97 Hs.99915 Androgen Receptor Up-regulated by Androgen Steroids9,531,96 Hs.2387 prostate-restricted Up-regulated by Androgen Biochem J315,901,96 transglutaminase Hs.78996 PCNA proliferating cellUp-regulated by Androgen Can Res 56,1539,96 nuclear antigen Hs.74456GAPDH Up-regulated by Androgen Can Res 55,4234,95 Hs.82004 E cadherinUp-regulated by Androgen BBRC, 212,624,95 Hs.57710 AIGF Androgen-inducedUp-regulated by Androgen FEBS lett 363,226,95 growth factor Hs.118618MIC2 humanpseudoautosom Up-regulated by Androgen Mol Carcinog, al gene?23,13,98 Hs.18420 Talin cytoskeletal protein Up-regulated by AndrogenFEBS lett 434,66,98 Hs.54502 clathrin heavy chain Up-regulated byAndrogen Endocrinology, 139,2111,98 Hs.73919 clathrin light chain bUp-regulated by Androgen Endocrinology, 139,2111,98 Hs.76506 L-plastinESTs, Moderately Up-regulated by Androgen Am J Pathol, 150, similar toL- 2009,97 PLASTIN [H. sapiens] Hs.82173 EGR alpha TGFB inducible earlyUp-regulated by Androgen Mol Endocrinol, growth response 9,1610,95 NDFGF10 Up-regulated by Androgen JBC, 274,12827,99 Hs.107169 IGFBP5Up-regulated by Androgen Endocrinology, 140,2372,99 Hs.179665 p21Up-regulated by Androgen Mol Endocrinol, 13,376,99 Hs.51117 BMP-7Up-regulated by Androgen Prostate, 37,236,98 Hs.73793 VEGF vascularendothelial Up-regulated by Androgen Endocrinol, 139,4672,98, growthfactor BBRC, 251,287,98 Hs.166 SREBPs sterol regulatory Up-regulated byAndrogen J Steroid Biochem Mol element binding Biol, 65,191,98transcription factor1 Hs.116577 PDF prostate Up-regulated by AndrogenJBC, 273,13760,98 differentiation factor Hs.1905 prolactin ProlactinUp-regulated by Androgen FEBS J, 11,1297,97 Hs.19192 CDK2 Up-regulatedby Androgen Can Res, 57,4511,97 Hs.95577 CDK4 cyclin-dependentUp-regulated by Androgen Can Res, 57,4511,97 kinase 4 Hs.183596 UGT2B17uridine Up-regulated by Androgen Endocrinology, diphosphoglucronosyl138,2998,97 transferase Hs.150207 UGT2B15 UDP- Up-regulated by AndrogenCan Res 57,4075,97 glucronosyltransferase 2B15 ND prostate bindingprotein Up-regulated by Androgen PNAS, 94,12999,97 C2A (RAT) ND Probasin(RAT) Up-regulated by Androgen PNAS, 94,12999,97 Hs.7719 prostatein C3(RAT) Up-regulated by Androgen PNAS, 94,12999,97 ND Cystatin relatedprotein 1 Up-regulated by Androgen PNAS, 94,12999,97 (RAT) ND Cystatinrelated protein 2 Up-regulated by Androgen PNAS, 94,12999,97 (RAT)Hs.394 Adrenomedulin (RAT) Up-regulated by Androgen PNAS, 94,12999,97Hs.77393 farnesyl diphosphate Up-regulated by Androgen PNAS, 94,12999,97synthase (farnesyl pyrophosphate synthetase,dimethylallyltranstransferase) Hs.153468 LDL receptor (Rat) Up-regulatedby Androgen PNAS, 94,12999,97 N.D. Hysto-blood group A Up-regulated byAndrogen PNAS, 94,12999,97 transferase (RAT) Hs.196604 Sex limitedprotein Up-regulated by Androgen PNAS, 94,12999,97 (RAT) slp NDprostatic spermine Up-regulated by Androgen Mol Cell Endocrinol, bindingprotein(RAT) 108, R1, 95 Hs.76353 Protein C Inhibitor Up-regulated byAndrogen FEBS lett, 492,263,98 Hs.203602 enolase alpha Up-regulated byAndrogen Can Res, 58,5718,98 Hs.169476 tubulin alpha Up-regulated byAndrogen Can Res, 58,5718,98 Hs.184572 Cdk1 Up-regulated by Androgen CanRes, 58,5718,98 Hs.107528 EST EST similar to Up-regulated by Androgenandrogen-regulated protein FAR-17 Hs.28309 UDP-glucose Up-regulated byAndrogen Endocrinology, dehydrogenase 140,10,4486,(99) Hs.194270secretory component Up-regulated by Androgen Mol endocrinol, gene13,9,1558,(99) Hs.76136 Thioredoxin Up-regulated by Androgen J steroidBiochem Mol Biol, 68, 5-6, 203, (99) Hs.3561 p27 Kip1 cyclin-dependentUp-regulated by Androgen Mol kinase inhibitor 1B Endocrinol, 12,941,98(p27, Kip1) Hs.1867 progastricsin Up-regulated by Androgen J.B.C.271,15175,(99) (pepsinogen C) Hs.97411 hamster Androgen- Up-regulated byAndrogen Genebank dependent Expressed Protein like protein geneHs.155140 Protein kinase CK2 casein kinase 2, alpha Translocated byAndrogen Can Res 59,1146,99 1 polypeptide IMAGE: 953262 DD3 ProstateSpecific Eur Urol, 35,408,99 Hs.218366 Prostase Prostate Specific PNAS,96,3114,99 Hs.20166 PSCA prostate stem cell Prostate Specific PNAS,95,1735,98 antigen Hs.171995 PSA kallikrein 3, (prostate ProstateSpecific PNAS, 95,300,98, specific antigen) DNA Cell Biol, 16,627,97Hs.183752 PSSPP prostate-secreted Prostate Specific PNAS, 95,300,98seminal plasma protein, nc50a10, microsemnoprotein beta, PSP94 Hs.1852PAP prostatic acid Prostate Specific PNAS, 95,300,98 phosphataseHs.52871 SYT Prostate Specific PNAS, 95,300,98 Hs.158309 Homeobox HOXD13 Prostate Specific PNAS, 95,300,98 Hs.1968 Semenogelin 1 ProstateSpecific PNAS, 95,300,98 Hs.76240 Adenylate kinase adenylate kinase 1Prostate Specific PNAS, 95,300,98 isoenzyme1 Hs.184376 SNAP23 ProstateSpecific PNAS, 95,300,98 Hs.82186 ERBB-3 receptor Prostate SpecificPNAS, 95,300,98 protein-tyrosin kinase Hs.180016 Semenogelin 2 ProstateSpecific Hs.1915 PSMA folate hydrolase Prostate Specific(prostate-specific membrane antigen) 1 Hs.181350 KLK2 Prostate SpecificHs.73189 NKX3.1 Prostate Specific HPARJ1 Prostate Specific IMAGE: 565779Hs.76053 p68 RNA helicase Potential interaction with AR MCB,19,5363,(99) Hs.111323 ARIP3 Potential interaction with AR JBC,274,3700,99 Hs.25511 ARA54 Potential interaction with AR JBC274,8319,99Hs.28719 ARA55 Potential interaction with AR JBC, 274,8570,99 HS. 999908ARA70 Potential interaction with AR PNAS, 93,5517,96 Hs.29131 TIF2transcriptional Potential interaction with AR EMBO, 15,3667,96,intermediary factor 2 EMBO, 17,507,98 Hs.66394 SNURF ring finger protein4 Potential interaction with AR MCB, 18,5128,98 Hs.75770 RBretinoblastoma 1 Potential interaction with AR (including osteosarcoma)Hs.74002 SRC-1 steroid receptor Potential interaction with ARcoactivator 1 Hs.155017 RIP140 nuclear receptor Potential interactionwith AR EMBO, 14,3741,95, interacting protein 1 Mol Endocrinol,12,864,98 Hs.23598 CBP CREB binding Potential interaction with ARprotein (Rubinstein- Taybi syndrome) Hs.25272 p300 E1A binding proteinPotential interaction with AR p300 Hs.78465 c-JUN Potential interactionwith AR Hs.199041 ACTR AIB1, mouse Potential interaction with AR M.C.B,17,2735,97, GRIP1, pCIP PNAS, 93,4948,96 Hs.6364 TIP60 Human tatinteractive Potential interaction with AR JBC, 274,17599,99 proteinmRNA, complete cds Hs.32587 SRA Potential interaction with AR Cell,97,17,99 Hs.155302 PCAF Potential interaction with AR Hs.10842 ARA24Potential interaction with AR Hs.41714 BAG-IL Potential interaction withAR JBC, 237,11660,98 Hs.82646 dnaJ, HSP40 DNAJ PROTEIN Potentialinteraction with AR HOMOLOG 1 Hs.43697 ERM ets variant gene 5 Potentialinteraction with AR JBC, 271,23907,96 (ets-related molecule) Hs.75772 GRPotential interaction with AR JBC, 272,14087,97 Hs.77152 MCM7 Potentialinteraction with AR ND NJ Potential interaction with AR ND RAF Potentialinteraction with AR JBC, 269,20622,94 ND TFIIF Potential interactionwith AR PNAS, 94,8485,97 Hs.90093 hsp70 Potential interaction with ARHs.206650 hsp90 Potential interaction with AR Hs.848 hsp56(FKBP52,Potential interaction with AR FKBP59, HBI)) Hs.143482Cyp40(cyclophilin40) Potential interaction with AR p23 Potentialinteraction with AR Hs.84285 ubiquitin-conjugating Potential Interactionwith AR J.B.C. 274,19441(99) enzyme Hs.182237 POU domain, class 2,Potential interaction with AR transcr Hs.1101 POU domain, class 2,Potential interaction with AR transcr Hs.2815 POU domain, class 6,Potential interaction with AR transcr IMAGE: 1419981 Potentialinteraction with AR Hs.227639 ARA160 Potential interaction with AR JBC,274,22373(99) Hs.83623 CAR-beta Xist locus Nuclear receptor gene familyHs.2905 PR Nuclear receptor gene family Hs.1790 MR mineralocorticoidNuclear receptor gene family receptor (aldosterone receptor) Hs.1657 ERalpha Nuclear receptor gene family Hs.103504 ER beta Nuclear receptorgene family Hs.110849 ERR1 Nuclear receptor gene family Hs.194667 ERR2Nuclear receptor gene family Hs.724 TR a thyroid hormone Nuclearreceptor gene family receptor, alpha (avian erythroblastic leukemiaviral (v-erb- a) oncogene homolog) Hs.121503 TR b Nuclear receptor genefamily Hs.171495 RAR b retinoic acid receptor, Nuclear receptor genefamily beta Hs.1497 RAR g retinoic acid receptor, Nuclear receptor genefamily gamma Hs.998 PPAR a Nuclear receptor gene family Hs.106415 PPAR bHuman peroxisome Nuclear receptor gene family proliferator activatedreceptor mRNA, complete cds Hs.100724 PPAR g peroxisome Nuclear receptorgene family proliferative activated receptor, gamma Hs.100221 LXR bNuclear receptor gene family Hs.81336 LXR a liver X receptor, Nuclearreceptor gene family alpha Hs.171683 FXR farnesoid X-activated Nuclearreceptor gene family receptor Hs.2062 VDR vitamin D (1,25- Nuclearreceptor gene family dihydroxyvitamin D3) receptor Hs.118138 PXR Nuclearreceptor gene family ND SXR Nuclear receptor gene family ND BXR Nuclearreceptor gene family ND CAR b? CAR a Nuclear receptor gene familyHs.196601 RXRA Nuclear receptor gene family Hs.79372 RXRB Human retinoidX Nuclear receptor gene family receptor beta (RXR- beta) mRNA, completecds Hs.194730?TR1? EAR1 Nuclear receptor gene family Hs.204704 EAR1 betaNuclear receptor gene family E75 Nuclear receptor gene family Hs.2156ROR alpha Nuclear receptor gene family Hs.198481 ROR beta Nuclearreceptor gene family Hs.133314 ROR gammma Nuclear receptor gene familyHs.100221 NER1 Nuclear receptor gene family Hs.54424 HNF4A Nuclearreceptor gene family Hs.202659 HNF4G Nuclear receptor gene familyHs.108301 TR2 Nuclear receptor gene family Hs.520 TR4 Nuclear receptorgene family Hs.144630 COUP-TF1 Nuclear receptor gene family Hs.1255COUP-TF2 Nuclear receptor gene family Hs.155286 EAR2 Nuclear receptorgene family Hs.1119 TR3 hormone receptor Nuclear receptor gene family(growth factor- inducible nuclear protein N10) Hs.82120 NURR1 IMMEDIATE-Nuclear receptor gene family EARLY RESPONSE PROTEIN NOT Hs.97196 SF1Nuclear receptor gene family Hs.183123 FTF fetoprotein-alpha 1 Nuclearreceptor gene family (AFP) transcription factor Hs.46433 DAX1 Nuclearreceptor gene family Hs.11930 SHP Homo sapiens nuclear Nuclear receptorgene family hormone receptor (shp) gene, 3′ end of cds Hs.83623,CAR-beta Nuclear receptor gene family IMAGE 1761923, or 1868028, or1563505, or 1654096 Hs.199078 Sin3 Nuclear receptor co-repressor complexNature, 387,43,97, Nature, 387,49,97 Hs.120980 SMRT Nuclear receptorco-repressor complex Nature, 377,454,95 Hs.144904 N-CoR Nuclear receptorco-repressor complex Nature, 377,297,95 Hs.188055 highly homologue geneNuclear receptor co-repressor complex to N-CoR in prostate and testisHs.180686 E6-AP Angelman syndrome Nuclear receptor co-activator complexMCB, 19,1182,99 associated protein Hs.199211?Hs. hBRM ESTs, Highlysimilar Nuclear receptor co-activator complex 198296? to HOMEOTIC GENEREGULATOR [Drosophila melanogaster] Hs.78202 hBRG1 Nuclear receptorco-activator complex Hs.11861 TRAP240 DRIP250, ARCp250 Nuclear receptorco-activator complex Mol Cell, 3,361,99 Hs.85313 TRAP230 ARCp240,DRIP240 Nuclear receptor co-activator complex Mol Cell, 3,361,99Hs.15589 TRAP220 RB18A, PBP, Nuclear receptor co-activator complexCRSP200, TRIP2, ARCp205, DRIP205 Hs.21586 TRAP170 RGR, CRSP150, Nuclearreceptor co-activator complex DRIP150, ARCp150 chromosomeX Hs.108319TRAP150 ESTs Nuclear receptor co-activator complex Mol Cell, 3,361,99Hs.193017 CRSP133 ARCp130, DRIP130 Nuclear receptor co-activator complexNature, 397,6718,99 Hs.23106 TRAP100 ARCp100, DRIP100, Nuclear receptorco-activator complex ND DRIP97 TRAP97 Nuclear receptor co-activatorcomplex Hs.24441 TRAP95 ESTs Nuclear receptor co-activator complex MolCell, 3,361,99 ND TRAP93 Nuclear receptor co-activator complex Hs.31659DRIP92 ARCp92? Nuclear receptor co-activator complex Hs.22630 TRAP80ARCp77, Nuclear receptor co-activator complex Mol Cell, 3,361,99 CRSP77,DRIP80(77)? Hs.204045 ARCp70 CRSP70, DRIP70 Nuclear receptorco-activator complex ND ARCp42 Nuclear receptor co-activator complex NDARCp36 Nuclear receptor co-activator complex Hs.184947 MED6 ARCp33Nuclear receptor co-activator complex Mol Cell, 3,97,99 Hs.7558 MED7CRSP33, ARCp34, Nuclear receptor co-activator complex Nature,397,6718,99 DRIP36 ND ARCp32 Nuclear receptor co-activator complex NDSRB10 Nuclear receptor co-activator complex ND SRB11 Nuclear receptorco-activator complex ND MED10 NUT2 Nuclear receptor co-activator complexHs.27289 SOH1 (yeast?) Nuclear receptor co-activator complex Mol Cell,3,97,99 ND p26 Nuclear receptor co-activator complex ND p28 Nuclearreceptor co-activator complex ND p36 Nuclear receptor co-activatorcomplex ND p37 Nuclear receptor co-activator complex ND but 2 TRFP humanhomologue of Nuclear receptor co-activator complex IMAGE clonesDrosophila TRF proximal protein ND VDR interacting subunit 180 kDa, HATNuclear receptor co-activator complex Genes Dev, 12,1787,98 activityHs.143696, or Coactivator associated Nuclear receptor co-activatorcomplex Science, 284,2174,99 IMAGE: 23716 methyltransferase 1 96?Hs.79387 SUG1 TRIP1 Nuclear receptor co-activator complex EMBO,15,110,96 ND TRUP Nuclear receptor co-activator complex PNAS, 92,9525,95Hs.28166 CRSP34 Nuclear receptor co-activator complex Nature,397,6718,99 Hs.63667 transcriptional adaptor 3 Nuclear receptorco-activator complex (A Hs.196725 ESTs, Highly similar to Nuclearreceptor co-activator complex P300 Hs.131846 PCAF associated factorNuclear receptor co-activator complex 65 al Hs.155635 ESTs, ModeratelyNuclear receptor co-activator complex similar to PCAF associated factor65 beta Hs.26782 PCAF associated factor Nuclear receptor co-activatorcomplex 65 beta Hs.118910 tumor suscitibility Modifying AR functionCancer 15,86,689, protein 101 (99) Hs.82932 Cyclin D1 cyclin D1 (PRADI:Modifying AR function Can Res, 59,2297,99 parathyroid adenomatosis 1)Hs.173664 HER2/Neu v-erb-b2 avian Modifying AR function PNAS, 9,5458,99erythroblastic leukemia viral oncogene homolog 2 Hs.77271 PKA proteinkinase, Modifying AR function JBC 274,7777,99 cAMP-dependent, catalytic,alpha Hs.85112 IGF1 insulin-like growth Modifying AR function Can Res,54,5474,94 factor 1 (somatomedin C) Hs.2230 EGF Modifying AR functionCan Res, 54,5474,94 Hs.129841 MEKK1 MAPKKK1 Modifying AR function MolCell Biol, 19,5143,99 Hs.83173 Cyclin D3 Modifying AR function Can Res,59,2297,99 Hs.75963 IGF2 Modifying AR function Hs.89832 InsulinModifying AR function Hs.115352 GH Modifying AR function Hs.1989 5 alphareductase type2 Involved in Androgen metabolism Hs.76205 CytochromeP450, Involved in Androgen metabolism subfamily XIA Hs.1363 CytochromeP450, Involved in Androgen metabolism subfamily XVII, (steroid17-alpha-hydroxylase), Hs.477 Hydroxysteroid (17- Involved in Androgenmetabolism beta) dehydrogenase 3 Hs.75441 Hydroxysteroid (17- Involvedin Androgen metabolism beta) dehydrogenase 4 Hs.38586Hydroxy-delta-5-steroid Involved in Androgen metabolism dehydrogenase, 3beta- and steroid delta- isomerase 1 Hs.46319 Sex hormone-bindingInvolved in Androgen metabolism globulin Hs.552 SRD5A1 Involved inAndrogen metabolism Hs.50964 C-CAM epithelial cell Down-regulated byAndrogen Oncogene, 18,3252,99 adhesion molecule Hs.7833 hSP56 seleniumbinding Down-regulated by Androgen Can Res, 58,3150,98 protein Hs.77432EGFR epidermal growth Down-regulated by Androgen Endocrinology, factorreceptor 139,1369,98 Hs.1174 p16 Down-regulated by Androgen Can Res,57,4511,97 Hs.55279 maspin Down-regulated by Androgen PNAS, 94,5673,97Hs.75789 TDD5 (mouse) Human mRNA for Down-regulated by Androgen PNAS,94,4988,97 RTP, complete cds Hs.75106 TRPM-2 clusterin ( Down-regulatedby Androgen testosterone-repressed prostate message 2, apolipoprotein J)Hs.25640 rat ventral prostate gene 1 claudin3 Down-regulated by AndrogenPNAS, 94,12999,97 ND glutathione S-transferase Down-regulated byAndrogen PNAS, 94,12999,97 Hs.25647 c-fos v-fos FBJ murineDown-regulated by Androgen PNAS, 94,12999,97 osteosarcoma viral oncogenehomolog N.D. matrix carboxyglutamic Down-regulated by Androgen PNAS,94,12999,97 acid protein (RAT) Hs.2962 S100P calcium bindingDown-regulated by Androgen Prostate 29,350,96 prottein Hs.75212ornithine decarboxilase ornithine Down-regulated by Androgen J Androl,19,127,98 decarboxylase 1 Hs.84359 Androge withdrawal Down-regulated byAndrogen apoptosis RVP1 Hs.79070 c-myc v-myc avian Down-regulated byAndrogen myelocytomatosis viral oncogene homolog Hs.139033 partiallyexpressed gene 3 Down-regulated by Androgen Mol Cell Endocrinol155,69,(99) Hs.20318 PLU-1 Associated with Prostate Cancer JBC,274,15633.99 Hs.18910 POV1(PB39) unique Associated with Prostate CancerGenomics, 51,282,98 Hs.119333 caveolin Associated with Prostate CancerClin Can Res, 4, 1873,98 ND, but 1 EST R00540(2.6 kbp) = 1M Associatedwith Prostate Cancer Urology, 50,302,97 IMAGE AGE: 123822 CLONEHs.184906 PTI-1 prostate tumor Associated with Prostate Cancer Can Res,57,18,97, inducing gene, PNAS, 92,6778,95 trancated and mutated humanelongation factor 1 alpha Hs.74649 cytochrome c oxidase Associated withProstate Cancer Can Res, 56,3634,96 subunit VI c Hs.4082 PCTA-1 prostatecarcinoma Associated with Prostate Cancer PNAS, 92,7252,96 tumorantigen, galectin family ND pp32r1 Associated with Prostate CancerNature Medicine, 5,275,99 ND pp32r2 Associated with Prostate CancerNature Medicine, 5,275,99 Hs.184945 GBX2 Associated with Prostate CancerThe prostate journal, 1,61,99 Hs.8867 Cyr61 inmmediate early Associatedwith Prostate Cancer Prostate, 36,85,98 protein Hs.77899 epithelialtropomyosin actin binding protein Associated with Prostate Cancer CanRes, 56,3634,96 Hs.76689 pp32 Associated with Prostate Cancer NatureMedicine, 5,275,99 Hs.10712 PTEN Associated with Prostate CancerHs.194110 KAI1 Associated with Prostate Cancer Hs.37003 H-ras Associatedwith Prostate Cancer Hs.184050 K-ras Associated with Prostate CancerHs.69855 N-ras neuroblastoma RAS Associated with Prostate Cancer viral(v-ras) oncogene homolog Hs.220 TGFbeta receptor1 Associated withProstate Cancer Hs.77326 IGFBP3 insulin-like growth Associated withProstate Cancer factor binding protein 3 Hs.79241 bcl-2 Associated withProstate Cancer Hs.159428 Bax Associated with Prostate Cancer Hs.206511bcl-x Associated with Prostate Cancer Hs.86386 mcl-1 myeloid cellleukemia Associated with Prostate Cancer sequence 1 (BCL2- related)Hs.1846 p53 tumor protein p53 Associated with Prostate Cancer(Li-Fraumeni syndrome) Hs.38481 CDK6 cyclin-dependent Associated withProstate Cancer kinase 6 Hs.118630 Mxi.1 Associated with Prostate CancerHs.184794 GAGE7 Associated with Prostate Cancer Hs.118162 fibronectinAssociated with Prostate Cancer Am J Pathol 154,1335,99 Hs.128231 PAGE-1Associated with Prostate Cancer JBC, 237,17618,98 Hs.75875 UEV1ubiquitin-conjugating Associated with Prostate Cancer Am J Pathol enzymeE2 variant 1 154,1335,99 Hs.75663 PM5 Human mRNA for Associated withProstate Cancer Am J Pathol pM5 protein 154,1335,99 Hs.180842 BBC1breast basic Associated with Prostate Cancer Am J Pathol conserved gene154,1335,99 Hs.198024 JC19 Associated with Prostate Cancer Can Res57,4075,97 N.D. GC79 novel gene Associated with Prostate Cancer Can Res57,4075,97 Hs.77054 B cell translocation gene 1 Associated with ProstateCancer Can Res 57,4075,97 Hs.78122 Regulatory factor X- Associated withProstate Cancer associated ankyrin- containing protein Hs.3337transmembranc 4 Associated with Prostate Cancer superfamily member1Hs.76698 TL5 Associated with Prostate Cancer Genebank Hs.3776 TL7Associated with Prostate Cancer Genebank Hs.170311 TL35 Associated withProstate Cancer Genebank Hs.184914 Human mRNA for T1- Associated withProstate Cancer 227H Hs.62954 ferritin, heavy Associated with ProstateCancer polypeptide Hs.71119 N33 Associated with Prostate CancerGenomics, 35,45(96)

[0261] TABLE 6 Genes/ESTs as defined by publications: Differentiallyexpresed genes in prostate cancer from CGAP database (NIH) Cluster.IDGene name Hs.179809 EST Hs.193841 EST Hs.99949 prolactin-induced proteinHs.101307 EST Hs.111256 arachidonate 15-lipoxygenase Hs.185831 ESTHs.115173 EST Hs.193988 EST Hs.159335 EST Hs.191495 EST Hs.187694 ESTHs.191848 EST Hs.193835 EST Hs.191851 EST Hs.178512 EST Hs.222886 ESTHs.210752 EST Hs.222737 EST Hs.105775 EST Hs.115129 EST Hs.115671 ESTHs.116506 EST Hs.178507 EST Hs.187619 EST Hs.200527 EST Hs.179736 ESTHs.140362 EST Hs.209643 EST Hs.695559 EST Hs.92323 MAT8 Hs.178391 BTKHs.55999 EST Hs.171185 Desmin Hs.54431 SGP28 Hs.182624 EST Hs.112259 Tcell receptor gammma Hs.76437 EST Hs.104215 EST Hs.75950 MLCK Hs.154103LIM Hs.9542 JM27 Hs.153179 FABP5 Hs.195850 EST Hs.105807 EST Hs.115089EST Hs.116467 EST Hs.222883 EST

[0262] TABLE 7 Androgen regulated Genes Defined by CPDR Genes/ESTsDerived from CPDR-Genome Systems ARG Database Cluster Gene NameDescription Hs.152204 TMPRSS2 Up-regulated by Androgen Hs.123107 KLK1Up-regulated by Androgen Hs.173334 elongation factor ell2 Up-regulatedby Androgen Hs.151602 epithelial V-like antigen Up-regulated by AndrogenHs.173231 IGFRI Up-regulated by Androgen Hs.75746 aldehyde dehydrogenase6 Up-regulated by Androgen Hs.97708 EST prostate and testis Up-regulatedby Androgen Hs.94376 proprotein convertase subtilisin/kexin type 5Up-regulated by Androgen AF017635 Homo sapiens Ste-20 related kinaseSPAK mRNA, complete cds {Incyte PD: Up-regulated by Androgen 60737}Hs.2798 leukemia inhibitory factor receptor Up-regulated by AndrogenHs.572 orosomucoid 1 Up-regulated by Androgen Hs.35804 KIAA0032 geneproduct Up-regulated by Androgen Hs.114924 solute carrier family 16(monocarboxylic acid transporters), member 6 Up-regulated by AndrogenHs.37096 zinc finger protein 145 (Kruppel-like, expressed inpromyelocytic leukemia) Up-regulated by Androgen R07295 sterolO-acyltransferase (acyl-Coenzyme A: cholesterol acyltransferase) 1Up-regulated by Androgen {Incyte PD: 2961248} Hs.118993-hydroxy-3-methylglutaryl-Coenzyme A reductase Up-regulated by AndrogenHs.216958 Human mRNA for KIAA0194 gene, partial cds Up-regulated byAndrogen Hs.76901 for protein disulfide isomerase-related Up-regulatedby Androgen Hs.180628 dynamin-like protein Up-regulated by AndrogenHs.81328 nuclear factor of kappa light polypeptide gene enhancer inB-cells inhibitor, Up-regulated by Androgen alpha Hs.159358acetyl-Coenzyme A carboxylase alpha Up-regulated by Androgen N24233IMAGE: 262457 Up-regulated by Androgen Hs.188429 EST Up-regulated byAndrogen Hs.77508 glutamate dehydrogenase 1 Up-regulated by AndrogenHs.12017 Homo sapiens KIAA0439 mRNA Up-regulated by Androgen Hs.10494EST Up-regulated by Androgen Hs.20843 EST Up-regulated by AndrogenHs.153138 origin recognition complex, subunit 5 (yeast homolog)-likeUp-regulated by Androgen Hs.79136 Human breast cancer, estrogenregulated LIV-1 protein (LIV-1) mRNA, partial Up-regulated by Androgencds Hs.35750 anthracycline resistance-associated Up-regulated byAndrogen Hs.56729 lymphocyte-specific protein 1 Up-regulated by AndrogenHs.17631 EST Up-regulated by Androgen Hs.46348 bradykinin receptor B1Up-regulated by Androgen Hs.172851 arginase, type II Up-regulated byAndrogen Hs.66744 twist (Drosophila) homolog Up-regulated by AndrogenHs.185973 membrane fatty acid (lipid) desaturase Up-regulated byAndrogen Hs.26 ferrochelatase (protoporphyria) Up-regulated by AndrogenHs.169341 ESTs, Weakly similar to phosphatidic acid phosphohydrolasetype-2c Up-regulated by Androgen [H. sapiens] Hs.119007 S-phase response(cyclin-related) Up-regulated by Androgen Hs.76285 H. sapiens gene fromPAC 295C6, similar to rat PO44 Up-regulated by Androgen Hs.167531 Homosapiens mRNA full length insert cDNA clone EUROIMAGE 195423 Up-regulatedby Androgen Hs.9817 arg/Abl-interacting protein ArgBP2 Up-regulated byAndrogen Hs.28241 EST Down-regulated by Androgen Hs.25925 Homo sapiensclone 23860 mRNA Down-regulated by Androgen Hs.10319 UDPglycosyltransferase 2 family, polypeptide B7 Down-regulated by AndrogenHs.155995 Homo sapiens mRNA for KIAA0643 protein, partial cdsDown-regulated by Androgen Hs.23552 EST Down-regulated by AndrogenHs.41693 DnaJ-like heat shock protein 40 Down-regulated by AndrogenHs.90800 matrix metalloproteinase 16 (membrane-inserted) Down-regulatedby Androgen Hs.2996 sucrase-isomaltase Down-regulated by AndrogenHs.166019 regulatory factor X, 3 (influences HLA class II expression)Down-regulated by Androgen Hs.27695 midline 1 (Opitz/BBB syndrome)Down-regulated by Androgen Hs.183738 chondrocyte-derived ezrin-likeprotein Down-regulated by Androgen Hs.75761 SFRS protein kinase 1Down-regulated by Androgen Hs.197298 NS1-binding protein Down-regulatedby Androgen Hs.149436 kinesin family member 5B Down-regulated byAndrogen Hs.81875 growth factor receptor-bound protein 10 Down-regulatedby Androgen Hs.75844 ESTs, Weakly similar to (defline not available5257244) [H. sapiens] Down-regulated by Androgen Hs.30464 cyclin E2Down-regulated by Androgen Hs.198443 inositol 1,4,5-triphosphatereceptor, type 1 Down-regulated by Androgen Hs.177959 a disintegrin andmetalloproteinase domain 2 (fertilin beta) Down-regulated by AndrogenHs.44197 Homo sapiens mRNA; cDNA DKFZp564D0462 (from cloneDown-regulated by Androgen DKFZp564D0462) Hs.150423 cyclin-dependentkinase 9 (CDC2-related kinase) Down-regulated by Androgen Hs.78776 Humanputative transmembrane protein (nma) mRNA, complete cds Down-regulatedby Androgen Hs.25740 ESTs, Weakly similar to !!!! ALU SUBFAMILY SQWARNING ENTRY !!!! Down-regulated by Androgen [H. sapiens] Hs.131041 ESTDown-regulated by Androgen Hs.19222 ecotropic viral integration site 1Down-regulated by Androgen Hs.9879 EST Down-regulated by AndrogenHs.118722 fucosyltransferase 8 (alpha (1,6) fucosyltransferase)Down-regulated by Androgen Hs.47584 potassium voltage-gated channel,delayed-rectifier, subfamily S, member 3 Down-regulated by AndrogenHs.115945 mannosidase, beta A, lysosomal Down-regulated by AndrogenHs.171740 ESTs, Weakly similar to Zic2 protein [M. musculus]Down-regulated by Androgen Hs.32970 signaling lymphocytic activationmolecule Down-regulated by Androgen Hs.196349 EST Down-regulated byAndrogen Hs.182982 Homo sapiens mRNA for KIAA0855 protein, partial cdsDown-regulated by Androgen Hs.72918 small inducible cytokine A1 (I-309,homologous to mouse Tca-3) Down-regulated by Androgen Hs.84232transcobalamin II; macrocytic anemia Down-regulated by Androgen Hs.10086EST Down-regulated by Androgen Hs.1327 ButyrylcholinesteraseDown-regulated by Androgen Hs.166684 serine/threonine kinase 3 (Ste20,yeast homolog) Down-regulated by Androgen AA558631 EST Down-regulated byAndrogen Hs.150403 dopa decarboxylase (aromatic L-amino aciddecarboxylase) Down-regulated by Androgen Hs.177548 postmeioticsegregation increased (S. cerevisiae) 2 Down-regulated by Androgen

[0263] TABLE 8 Other Genes Associated with Cancers Cluster Gene nameDescription Hs.146355 c-Abl v-abl Abelson murine leukemia viral oncogenehomolog 1 Hs.96055 E2F1 Hs.170027 MDM2 Hs.1608 RPA replication proteinA3 (14 kD) Hs.99987 XPD ERCC2 Hs.77929 XPB ERCC3 Hs.1100 TBP TATA boxbinding protein Hs.60679 TAFII31 TATA box binding protein(TBP)-associated factor, RNA polymerase II, G, 32 kD Hs.78865 TAFII70Human TBP-associated factor TAFII80 mRNA, complete cds Hs.178112 DP1deleted in poliposis Hs.119537 p62 Hs.48576 CSB excision repaircross-complementing rodent repair deficiency, complementation group 5Hs.73722 Ref-1 Hs.194143 BRCA1 breast cancer 1, early onset Hs.184760CBF Hs.1145 WT-1 Wilms tumor 1 Hs.2021 Sp1 Hs.144477 CK I Hs.155627DNA-PK Hs.170263 p53BP1 Human clone 53BP1 p53-binding protein mRNA,partial cds Hs.44585 p53BP2 tumor protein p53-binding protein, 2 Hs.6241p85 alpha PI3 kinase Hs.23707 p85 beta PI3 kinase Hs.194382 ATMHs.184948 BIN1 Hs.137569 p51B p63 Hs.1334 bmyb v-myb avianmyeloblastosis viral oncogene homolog Hs.81942 DNA polymerase polymerase(DNA directed), alpha alpha Hs.180952 Beta actin Hs.93913 IL-6interleukin 6 (interferon, beta 2) Hs.190724 MAP4 microtubule-associatedprotein 4 Hs.1384 MGMT o-6-methylguanine-DNA methyltransferase Hs.79572Cathepsin D cathepsin D (lysosomal aspartyl protease) Hs.111301Collagenase IV Hs.151738 Collagenase IV Hs.51233 DR5 Hs.82359 FASHs.80409 GADD45 DNA-damage-inducible transcript 1 Hs.86161 GMLGPI-anchored molecule like protein Hs.50649 PIG3 quinone oxidoreductasehomolog Hs.184081 Siah seven in absentia (Drosophila) homolog 1 Hs.56066bFGF fibroblast growth factor 2 (basic) Hs.205902 IGF1-R Hs.21330 MDR1 Pglycoprotein 1/multiple drug resistance 1 Hs.74427 PIG11 Homo sapiensPig11 (PIG11) mRNA, complete cds Hs.76507 PIG7 LPS-induced TNF-alphafactor Hs.8141 PIG8 Hs.146688 PIG12 Hs.104925 PIG10 Hs.202673 PIG6Hs.80642 STAT4 Hs.72988 STAT2 Hs.167503 STAT5A Hs.738 early growthresponse 1 Hs.85148 villin2 Hs.109012 MAD Hs.75251 DEAD/H box bindingprotein 1 Hs.181015 STAT6 Hs.199791 SSI-3 STAT induced STAT inhibitor 3Hs.21486 STAT1 Hs.142258 STAT3 Hs.76578 PIAS3 Protein inhibitor ofactivated STAT3 Hs.44439 CIS4 STAT induced STAT inhibitor 4 Hs.50640SSI-1 JAK binding protein Hs.54483 NMI N-Myc and STAT interactorHs.105779 PIASy Protein inhibitor of activated STAT Hs.110776 STATI2STAT induced STAT inhibitor 2 Hs.181112 EST similar to STAT5A

[0264] TABLE 9 Functional Categories of ARGs Tag T/C Access # Name,Description Transcription Regulators GCCAGCCCAG (SEQ ID NO: 13) 11/1 H41030 KAP1/TIF1beta, KRAB-associated protein 1 GTGCAGGGAG (SEQ ID NO:14) 18/2  AF071538 PDEF, ets transcription factor GACAAACATT (SEQ ID NO:15) 8/1 NM_003201 mtTF1, mitochondrial transcription factor 1 ATGACTCAAG(SEQ ID NO: 16) 8/1 X12794 ear-2, v-erbA related GAAAAGAAGG (SEQ ID NO:17) 8/1 U80669 Nkx3.1, homeobox CCTGTACCCC (SEQ ID NO: 18) 5/1 AF072836Sox-like transcriptional factor CCTGAACTGG (SEQ ID NO: 19) 1/8 NM_001273CHD4/Mi2-beta, histone acetylase/deacetylase, chromodomain helicaseTGACAGCCCA (SEQ ID NO: 20) 1/7 U81599 Hox B13, homeobox RNA Processingand Translational Regulators TACAAAACCA (SEQ ID NO: 21) 12/1  NM_005381NCL, Nucleolin AATTCTCCTA (SEQ ID NO: 22) 8/1 U41387 GURDB, nucleolarRNA helicase TGCATATCAT (SEQ ID NO: 23) 8/1 D89729 XPO1, exportin 1CTTGACACAC (SEQ ID NO: 24) 14/2  AL080102 EIF5, translation initiationfactor 5 TGTCTAACTA (SEQ ID NO: 25) 5/1 AF078865 CGI-79, RNA-bindingprotein GTGGACCCCA (SEQ ID NO: 26) 10/2  AF190744 SiahBP1/PUF60, poly-Ubinding splicing factor ATAAAGTAAC (SEQ ID NO: 27)  1/11 NM_007178UNRIP, unr-interacting protein. TACATTTTCA (SEQ ID NO: 28) 1/7 X85373SNRPG, small nuclear RNP polypeptide G TCAGAACAGT (SEQ ID NO: 29) 1/7NM_002092 GRSF-1, G-rich RNA binding factor 1 CAACTTCAAC (SEQ ID NO: 30)0/5 NM_006451 PAIP1, poly A BP-interacting protein 1 GATAGGTCGG (SEQ IDNO: 31) 0/5 Z11559 IREBP1, Iron-responsive element BP 1 CTAAAAGGAG (SEQID NO: 32)  2/10 M15919 SNRPE, small nuclear RNP polypeptide E GenomicMaintenance and Cell Cycle Regulation GTGGTGCGTG (SEQ ID NO: 33) 10/1 AF035587 XRCC2, X-ray repair protein 2 TCCCCGTGGC (SEQ ID NO: 34) 7/1D13643 KIAA0018, Dimunuto-like ATTGATCTTG (SEQ ID NO: 35) 6/1 NM_002947RPA3, Replication protein A 14kDa subunit AGCTGGTTTC (SEQ ID NO: 36)16/3  NM_004879 PIG8, p53 induced protein CCTCCCCCGT (SEQ ID NO: 37)10/2  AF044773 BAF, barrier-to-autointegration factor ATGTACTCTG (SEQ IDNO: 38) 1/7 NM_000884 IMPDH2, IMP dehydrogenase 2 GATCAAATAC (SEQ ID NO:39) 0/5 NM_006325 ARA24, androgen receptor assoc protein 24 GTGCATCCCG(SEQ ID NO: 40) 0/5 X16312 Phosvitin/casein kinase II beta subunitProtein Trafficking and Chaperoning GAAATTAGGG (SEQ ID NO: 41) 12/1 AB020637 KIAA0830, similar to golgi antigen TTTCTAGGGG (SEQ ID NO: 42)10/1  AF15189 CGI-140, lysosomal alpha B mannosidase CCCAGGGAGA (SEQ IDNO: 43) 7/1 AF026291 CCT, chaperonin t-complex polypeptide 1 GTGGCGCACA(SEQ ID NO: 44) 13/2  S79862 26 S protease subunit 5b TTGCTTTTGT (SEQ IDNO: 45) 15/3  NM_001660 ARF4, ADP-ribosylation factor 4 ATGTCCTTTC (SEQID NO: 46) 10/2  NM_005570 LMAN1, mannose BP involved in EPR/Golgitraffic Energy Metabolism, Apoptosis and Redox Regulators TGTTTATCCT(SEQ ID NO: 47) 13/2  M14200 DBI, diazepam binding inhibitor GCTTTGTATC(SEQ ID NO: 48) 6/1 D16373 dihydrolipoamide succinyltransferaseGTTCCAGTGA (SEQ ID NO: 49) 6/1 AA653318 FKBP5, FK506-binding protein 5TAGCAGAGGC (SEQ ID NO: 50) 6/1 AA425929 NDUFB10, NADH dehydrogenase 1beta subcomplex 10 ACAAATTATG (SEQ ID NO: 51) 5/1 NM_003375 VDAC,voltage-dependent anion channel CAGTTTGTAC (SEQ ID NO: 52) 5/1 NM_000284PDHA1, Pyruvate dehydrogenase E1-alpha subunit GATTACTTGC (SEQ ID NO:53) 5/1 NM_004813 PEX16, peroxisomal membrane biogenesis factorGGCCAGCCCT (SEQ ID NO: 54) 5/1 X15573 PFKL, 1-phosphofructokinaseCAATTGTAAA (SEQ ID NO: 55)  1/10 NM_004786 TXNL, thioredoxin-likeprotein AAAGCCAAGA (SEQ ID NO: 56)  2/15 NM_001985 ETFB, electrontransfer flavoprotein beta subunit CAACTAATTC (SEQ ID NO: 57) 1/7NM_001831 CLU, Clustrin AAGAGCTAAT (SEQ ID NO: 58) 0/5 NM_004446 EPRS,glutamyl-prolyl-tRNA synthetase Signal Transduction CTTTTCAAGA (SEQ IDNO: 59) 9/1 X59408 CD46, complement system membrane cofactor GTGTGTAAAA(SEQ ID NO: 60) 9/1 NM_005745 BAP31/BAP29 IgD accessory proteinsACAAAATGTA (SEQ ID NO: 61) 8/1 NM_000856 GUCY1A3, Guanylate cyclase 1,alpha 3 AAGGTAGCAG (SEQ ID NO: 62) 7/1 NM_006367 CAP, Adenylylcyclase-associated protein GGCGGGGCCA (SEQ ID NO: 63) 7/1 AB002301microtubule assoc. serine/threonine kinase GGCCAGTAAC (SEQ ID NO: 64)6/1 AL096857 similar to BAT2, integrin receptor AACTTAAGAG (SEQ ID NO:65) 12/2  AB018330 calmodulin-dependent protein kinase kinase βAGGGATGGCC (SEQ ID NO: 66) 5/1 NM_006858 IL1RL1LG, Putative T1/ST2receptor CTTAAGGATT (SEQ ID NO: 67)  2/10 AF151813 CGI-55 protein

[0265] The “tag to gene” identification is based on the analysisperformed by SAGE software and/or “tag to gene” application of the NIHSAGE Website. T/C represent the number of tags for each transcript inandrogen treated (T) and control (C) LNCaP libraries. The differences inexpression levels of genes identified by tags shown here werestatistically significant (p<0.05) as determined by the SAGE software.

REFERENCES

[0266] 1. Landis S H, Murray T, Bolden S, and Wingo P A: Cancerstatistics. CA Cancer J. Clin., 49:8-31, 1999.

[0267] 2. Pannek J and Partin A W: Prostate-specific Antigen: What isnew in 1977. Oncology 11, 1273-1282, 1997.

[0268] 3. Small E J: Update on the diagnosis and treatment of prostatecancer: Curr. Opin. Oncol., 10:244-252, 1998.

[0269] 4. Krongrad A, Lai H, and Lai S: Survival after radicalprostatectomy. JAMA, 278:44-46, 1997.

[0270] 5. Garwick, M B and Fair W R: Prostate Cancer, ScientificAmerican, 75-83, 1998.

[0271] 6. Augustus M, Moul J W, and Srivastava S: The molecularphenotype of the malignant prostate. Molecular pathology of early cancer(in press), 1999.

[0272] 7. Sakr W A, Macoska J A, Benson P, Benson D J, Wolman S R,Pontes J E, and Crissman: Allelic loss in locally metastatic,multi-sampled prostate cancer. Cancer Res., 54:3273-3277, 1994.

[0273] 8. Mirchandani D, Zheng J, Miller G L, Ghosh A K, Shibata D K,Cote R J and Roy-Burman P: Heterogeneity in intratumor distribution ofp53 mutations in human prostate cancer. Am. J. Path. 147:92-101, 1995.

[0274] 9. Bauer J J, Moul J W, and McLeod D G: CaP: Diagnosis,treatment, and experience at one tertiary medical center, 1989-1994.Military Medicine, 161:646-653,1996.

[0275] 10. Moul J W, Gaddipati J, and Srivastava S: 1994. Molecularbiology of CaP. Oncogenes and tumor suppressor genes. Current ClinicalOncology: CaP. (Eds. Dawson, N. A. and Vogelzang, N. J.), Wiley-LissPublications, 19-46.

[0276] 11. Lalani E-N, Laniado M E and Abel P D: Molecular and cellularbiology of prostate cancer. Cancer and Mets. Rev., 16:29-66, 1997.

[0277] 12. Shi X B, Gumerlock P H, deVere White R W: Molecular Biologyof CaP. World J. Urol; 14, 318-328, 1996.

[0278] 13. Heidenberg H B, Bauer J J, McLeod D G, Moul J W andSrivastava S: The role of p53 tumor suppressor gene in CaP: a possiblebiomarker? Urology, 48:971-979, 1996.

[0279] 14. Bova G S and Issacs W B: Review of allelic loss and gain inprostate cancer. World J Urol., 14:338-346, 1996.

[0280] 15. Issacs W B and Bova G S: Prostate Cancer: The Genetic Basisof Human Cancer. Eds. Vogelstein B, and Kinzler K W, McGraw-HillCompanies, Inc., pp. 653-660, 1998.

[0281] 16. Heidenberg H B, Sesterhenn I A, Gaddipati J, Weghorst C M,Buzard G S, Moul J W, and Srivastava S: Alterations of the tumorsuppressor gene p53 in a high fraction of treatment resistant prostatecancer. J. Urol., 154:414-421, 1995.

[0282] 17. Bauer J J, Sesterhenn I A, Mostofi F K, McLeod D G,Srivastava S, Moul J W: p53 protein expression is an independentprognostic marker in clinically localized prostate cancer patients.Clin. Cancer Res., 1:1295-1300, 1995.

[0283] 18. Bauer J J, Sesterhenn I A, Mostofi F K, McLeod D G,Srivastava S, Moul, J W: Elevated levels of apoptosis regulator proteinsp53 and bcl-2 are independent prognostic biomarkers in surgicallytreated clinically localized prostate cancer patients. J. Urol.,1511-1516,1996.

[0284] 19. Yang G, Stapleton A M, Wheeler T M, Truong L D, Timme T O,Scardino T P, and Thompson T O: Clustered p53 immunostaining. A novelpattern associated with prostate cancer progression. Clin. Cancer Res.,2:399-401, 1996.

[0285] 20. Cairns P, Okami K, Halachmi S, Halachmi N, Esteller M, HermanJ G, Jen J, Isaacs W B, Bova G S, and Sidransky D: Frequent inactivationof PTEN/MMAC1 in primary prostate cancer. Cancer Res, 57:4997-5000,1997.

[0286] 21. Suzuki H, Freije D, Nusskern D R, Okami K, Cairns P,Sidransky D, Isaacs W B, and Bova G S: Interfocal heterogeneity ofPTEN/MMAC 1 gene alterations in multiple metastatic prostate cancertissues: Cancer Res, 58:204-209, 1998.

[0287] 22. Jenkins R B, Qian J, Lieber M M and Bostwick D G: Detectionof c-myc oncogene amplification and chromosomal abnormalities inmetastatic prostatic carcinoma by fluorescence in situ hybridization.Cancer Res, 57:524-531, 1997.

[0288] 23. Reiter R E, Gu Z, Watabe T., Thomas G, Szigeti K, Davis E,Wahl M, Nisitani S, Yamashiro I, LeBeau M M, Loda M and Witte O N:Prostate stem cell antigen: a cell surface marker overexpressed inprostate cancer. Proc Natl Acad Sci, 95:1735-40, 1998.

[0289] 24. Visakorpi T, Kallioniemi A H, Syvanen A, Hyytinen E R, KarhuR, Tammela T, Isola J J and Kallioniemi O—P: Genetic changes in primaryand recurrent prostate cancer. Cancer Res, 55:342-347, 1995.

[0290] 25. Cher M L, Bova G S, Moore D H, Small E J, Carroll P A, Pinn SS, Epstein J L, Isaacs W B and Jensen R H: Genetic alterations inuntreated metastases and androgen-independent prostate cancer detectedby comparative genomic hybridization and allotyping. Cancer Res,56:3091-3102, 1996.

[0291] 26. Srikantan V, Sesterhenn I A, David L, Hankins G R, Avallone FA, Livezey J R, Connelly R, Mostofi F K, McLeod D G, Moul J W,Chandrasekharappa, S C, and Srivastava S: Chromosome 6q alterations inhuman prostate cancers. Int J Cancer (in press), 1999.

[0292] 27. Smith J R, Freije D, Carpten J D, Gronberg H, et al: Majorsusceptibility locus for prostate cancer on chromosome 1 suggested by agenome-wide search. Science, 276:1371-1374, 1996.

[0293] 28. Xu J, Meyers D, Freije D, Issacs S, et al: Evidence for aprostate cancer susceptibility locus on x chromosome. Nat. Genet, 20:175-179, 1998.

[0294] 29. Liang, Peng, and Pardee A B: Differential display ofeukaryotic messenger RNA by means of the polymerase chain reaction.Science 257:967-971, 1992.

[0295] 30. Velculescu V E, Zhang L, Vogelstein B, and Kinzler K W:Serial analysis of gene expression Science, 270:484-487, 1995.

[0296] 31. Chena M, Shalon D S, Davis R W, and Brown P O: Quantitativemonitoring of gene expression patterns with a complementary DNAmicroarrays. Science, 270:467-470, 1995.

[0297] 32. Srikantan V., Zou Z, Davis L D, Livezey J, Sesterhenn I A, XuL, Mostofi F K, McLeod D G, Moul J W, and Srivastava S: Structure andexpression of a novel prostate specific gene PCGEM1. American Assoc.Cancer Res. Meeting, Philadelphia, Pa., 1999.

[0298] 33. Xu, L, Su Y, Labiche R, McLeod D G, Moul J W and SrivastavaS: Probing the androgen regulated genes (ARGs) in prostate cancer cellsby serial analysis of gene expression (SAGE). American Assoc. of CancerResearch Meeting, 1999.

[0299] 34. Huggins, C., Hodges, C. V. Studies on prostate cancer,effects of castration, of estrogens and androgen injection on serumphosphatase in metastatic carcinoma of the prostate. Cancer Res,1:293-297,1941.

[0300] 35. Moul J W: Contemporary hormonal management of advancedprostate cancer. Oncology, 12: 499-505, 1998.

[0301] 36. Veldscholte, J, Ris-Stalpers C, Kulper G GJM, Jenster G,Berre-voets C, Classen E, Van Roooj H C J, Trapman J, Brinkmann A O,Mulder E. A mutation in the ligand binding domain of the androgenreceptor of human LNCaP cells affects steroid binding characteristicsand response to anti-androgens. Biochem. Biophys. Res. Commun.,173:534-540, 1990.

[0302] 37. Newmark J R, Hardy 0, Tonb D C, Carter B S, Epstein J I,Isaacs W B, Brown T R, Barrack E R. Androgen receptor gene mutations inhuman prostate cancer. Proc Natl Acad Sci USA, 89:6319-6323, 1992.

[0303] 38. Culig Z, Hobisch A, Cronauer M V, Cato A C B, Hittmair A,Radmayr C, Eberie J, Bartsch G, Klocker H. Mutant androgen receptordetected in an advanced stage prostatic carcinoma is activated byadrenal androgens and progesterone. Mol. Endocrinol, 7:1541-1550, 1993.

[0304] 39. Suzuki H, Sato N, Watabe Y, Masai M, Seino S, Shimazaki S.Androgen receptor gene mutations in human prostate cancer. J SteroidBiochem Mol Biol, 46:759-765, 1993.

[0305] 40. Gaddipatti J P, McLeod D G, heidenberg H B, Sesterhann I A,Finger M J, Moul J W, Srivastava S. Frequent detection of codon 877mutation in the androgen receptor gene in advanced prostate cancers.Cancer Res, 54:2861-2864.1994.

[0306] 41. Peterziel H, Culig Z, Stober J, Hobisch A, Radmayr C, BartschG, Klocker, Cato A C B. Mutant androgen receptors in prostate cancerdistinguish between amino acid sequence requirements for transactivationand ligand binding. Int J Cancer, 63:544-550, 1995.

[0307] 42. Taplin M-E, Bubley G J, Shuster T D, Frantz M E, Spooner A E,Ogata G K, Keer H N, Balk S P. Mutation of the androgen receptor gene inmetastatic androgen independent prostate cancer. N Engl J Med,332:1393-1398.

[0308] 43. Tilley W D, Buchanan G, Hickey T E, Bental J M. Mutation inthe androgen receptor gene are associated with progression of humanprostate cancer to androgen independence. Clin Cancer Res, 2:277-285,1994.

[0309] 44. Visakorpi T, Hyytinen E, Koivisto P, tanner M, Keinanen R,Palmberg C, Tammela T, Isola J, Kallioniemi O P. In vivo amplificationof the androgen receptor gene and progression of human prostate cancer.Nature Genet, 9:401-406, 1995.

[0310] 45. Koivisto P, Kononen J, Palmberg C, Tammela T, Hyytinen E,Isola J, Trapman J, Cleutjens K, Noordzij A, Visakorpi T, Kallioniemi OP. Androgen receptor gene amplification: a possible molecular mechanismfor androgen deprivation therapy failure in prostate cancer. Cancer Res,57:314-318, 1997.

[0311] 46. Culig Z, Hobisch A, Cronauer M V, Radmayr C, Trapman J,Hittmair A, Bartsch G, Klocker H. Androgen receptor activation inprostate tumor cell lines by insulin-like growth factor-1, keratinocytegrowth factor, and epidermal growth factor. Cancer Res, 54:5474-5478,1994.

[0312] 47. Yeh S, Chang C. Cloning and characterization of a specificcoactivator, ARA70, for the androgen recptor in human prostate cells.Proc Natl Acad Sci USA, 93:5517-5521,1996.

[0313] 48. Nagabhushan M, Miller C M, Pretlow T P, Giaconia J M,Edgehouse N L, Schwartz S, Kung H-J, deVere White R W, Gumerlock P H,Resnick M I, Amini S B, Pretlow T G. CWR22: The first human prostatecancer Xenograft with strongly androgen-independent and relapsed strainsboth in vivo and in soft agar. Cancer Res, 56:3402-4306, 1996.

[0314] 49. Gregory C W, Hamil K G, Kim D, Hatt S H, Pretlow T G, MohlerJ L, French F S. Androgen receptor expression in androgen independentcancer is associated with increased expression of androgen regulatedgenes. Cancer Res, 58:5718-5724,1998.

[0315] 50. Noble R L: The development of prostatic adenocarcinoma in Nbrats following prolonged sex hormone administration. Cancer Res,37:1929-1933,1977.

[0316] 51. Pollard M: Lobund-Wistar rat model of prostate cancer in man.Prostate, 37:1-4, 1998.

[0317] 52. Pollard M, Luckert P H, and Snyder D L: The promotionaleffect of testosterone on induction of prostate cancer in MNU-sensitizedL-W rats. Cancer Lett, 45:209-212, 1989.

[0318] 53. Gann P H, Hennekens C H, Ma J, Longcope C, Stampfer M J:Prospective study of sex hormone levels and risk of prostate cancer JNatl Cancer Inst, 88:1118-1126,1996.

[0319] 54. Hakimi J M, Schoenberg M P, Rondinelli R H, Piantadosi S,Barrack E R. Androgen receptor variants with short glutamine or glycinerepeats may identify unique subpopulations of men with prostate cancer.Clin Cancer Res, 9:1599-1608, 1997.

[0320] 55. Giovanucci E, Stampfer M J, Krithivas K, Brown M, Brafsky A,Talcott J, Hennekens C H, Kantoff P W. The CAG repeat within theandrogen receptor gene and its relationship to prostate cancer. ProcNatl Acad Sci USA, 94:3420-3423,1997.

[0321] 56. Coetzee G A, Ross R K. Prostate cancer and the androgenreceptor. J. Natl Cancer Inst, 86:872-873, 1994.

[0322] 57. Moul J W. Increased risk of prostate cancer in African men.Mol. Urol, 1:119-127,1997.

[0323] 58. Chamberlain N L, Driver E D, Miesfeld R L. The length andlocation of CAG trinucleotide repeats in the androgen receptorN-terminal domain affect transactivation function. Nucleic Acids Res,22:3181-3186, 1994.

[0324] 59. Trapman J, Cleutzens KBJM. Androgen regulated gene expressionin prostate cancer. Seminars in Canc Biol, 8:29-36,1997.

[0325] 60. Yuan S, Trachtenberg J, Mills G B, Brown T J, and Keating A:Androgen-induced inhibition of cell proliferation in anandrogen-insensitive prostate cancer cell line (PC3) transfected withhuman androgen receptor complementary DNA. Cancer Res, 53:1304-1311,1993.

[0326] 61. Velculescu V E, Zhang L, Vogelstein B, and Kinzler K W:Serial Analysis of Gene Expression. Science, 270, 484-487, 1995

[0327] 62. Polyak K, Yong X, Zweier J L, Kinzler K W, and Vogelstein B:A model for p53 induced apoptosis. Nature, 389, 300-306, 1997.

[0328] 63. Hermeking H, Lengauer C, Polyak C, He T-C, Zhang L,Thiagalingam S, Kinzler KW, and Vogelstein B: 14-3-3 is a p53-regulatedinhibitor of G2/M progression. Molecular Cell, 1: 3-11, 1997.

[0329] 64. He T-C, Sparks A B, Rago C, Hermeking H, Zawel L, da Costa LT, Morin P J, Vogelstein B, and Kinzler K W: Identification of c-myc asa target of the APC pathway. Science, 281;1438-1441, 1998.

[0330] 65. Bieberich, C. J., Fujita, K., He, W. W., and Jay, G.:Prostate-specific and androgen-dependent expression of a novel homeoboxgene. J Biol Chem, 271: 31779-31782, 1996.

[0331] 66. Sciavolino, P. J., Abrams, E. W., Yang, L., Austenberg, L.P., Shen, M. M., and Abate-Shen, C.: Tissue-specific expression ofmurine Nkx3.1 in the male urogenital sinus. Dev Dyn, 209: 127-138, 1997.

[0332] 67. He, W. W., Sciavolino, P. J., Wing, J., Augustus, M., Hudson,P., Meissner, S. P., Curtis, R. T., Shell, B. K., Bostwick, D. G.,Tindall, D. J., Gelmann, E. P., Abate-Shen, C., and Carter, K. C.: Anovel human prostate-specific androgen-regulated homeobox gene (NKX3.1)that maps to 8p21, a region frequently deleted in prostate cancer.Genomics, 43: 69-77, 1997.

[0333] 68. Prescott J. L., Blok L., and Tindall D. J.: Isolation andandrogen regulation of the human homeobox cDNA, NKX3.1. The Prostate,35: 71-80, 1998.

[0334] 69. Xu L, Srikantan V, Sesterhenn I A, Augustus M, Sui D, Moul JW, Carter KC and Srivastava S: Evaluation of expression of androgenregulated prostate specific homeobox gene, NKX3.1 in human prostatecancer. Int. Symp. on Biol. of Prostate Growth, Bethesda, 176, 1998;Manuscript submitted to J Urol, 1999.

[0335] 70. Voeller, H. J, Augustus, M, Madike, V., Bova, G. S., Carter,K. C., and Gelmann, E. P.: Coding region of NKX3.1, a prostate-specifichomeobox gene on 8p21, is not mutated in human prostate cancers. CancerRes, 57: 4455-4459, 1997.

[0336] 71. Song, K., Wang, Y., and Sassoon, D.: Expression of Hox-7.1 inmyoblasts inhibits terminal differentiation and induces celltransformation. Nature, 360: 477-481, 1992.

[0337] 72. Maulbecker, C. C., and Gruss, P.: The oncogenic potential ofderegulated homeobox genes. Cell Growth Differ, 4: 431-441,1993.

[0338] 73. Krosl, J., Baban, S., Krosl, G., Rozenfeld, S., Largman, C.,and Sauvageau, G.: Cellular proliferation and transformation induced byHOXB4 and HOXB3 proteins involves cooperation with PBX1. Oncogene, 16:3403-3412, 1998.

[0339] 74. Kaighn M E, Reddel R R, Lechner J F, Peehl D M, Camalier R F,Brash D E, Saffioti U, and Harris C C: Transformation of human neonatalprostate epithelial cells strontium phosphate transfection with plasmidcontaining SV40 early region genes. Cancer Res, 49: 3050-3056,1989.

[0340] 75. Kuettel M R, Thraves P J, Jung M, Varghese S P, Prasad S C,Rhim J S, and Dritschilo A: Radiation-induced neoplastic transformationof human prostate epithelial cells. Cancer Res, 56:5-10,1996.

[0341] 76. Srivastava S, Wheelock RHP, Eva A, and Aaronson S A:Identification of the protein encoded by novel human diffuse B celllymphoma oncogene. Proc Natl Acad Sci, USA, 83:8868-8872, 1986.

[0342] 77. Graziani G, Ron D, Eva A, and Srivastava: The human dblproto-oncogene product is a cytoplasmic phosphoprotein which isassociated with cytoskeletal matrix. Oncogene, 4:823-829, 1989.

[0343] 78. Srivastava S, Zou Z, Pirollo K, Blattner W, and Chang E S:Germ-line transmission of a mutated p53 gene in a cancer-prone familywith Li-Fraumeni syndrome. Nature, 348:747-749, 1990.

[0344] 79. Srivastava, S., Wang, S., Tong, Y. A., Hao, Z. M. and Chang,E. H.: Dominant negative effect of a germ-line mutant p53: a stepfostering tumorigenesis. Cancer Res, 53:4452, 1993.

[0345] 80. Gaddipati J P, Mcleod D G, Sesterhenn I A, Hussussian C J,Tong Y A, Seth P, Dracopoli N C, Moul J M, and Srivastava, S: Mutationsof p16 gene are rare in prostate cancer. Prostate, 30:188-194, 1997.

[0346] 81. Bonner R F, Emmert-Buck M, Cole K, Pohida T, Chuaqi R,Goldstein S, and Liotta L A: Laser capture microdissection: molecularanalysis of tissue. Science, 278:1481-1483, 1997.

[0347] Bastian, B. C., Le Boit, P. E., Hamm, H., Brocker, E. B., andPinkel, D. (1998). Chromosomal gains and losses in primary cutaneousmelanomas detected by comparative genomic hybridization. Cancer Res. 58:2170-2175.

[0348] Bentel, J. M., Tilley, W. D. (1996). Androgen receptors inprostate cancer. J Endocrinology 151: 1-11.

[0349] Brothman, A. R., Peehl, D. M., Patel, A. M., and McNeal, J. E.(1990). Frequency and pattern of karyotypic abnormalities in humanprostate cancer. Cancer Res. 50: 3795-3803.

[0350] Cuthill, S. (1999). Dominant genetic alterations inimmortalization: Role for 20q gain. Genes Chromosomes Cancer 26:304-311.

[0351] Gregory, C. W., Hamil, K. G., Kim, D., Hall, S. H., Pretlow, T.G., Mohler, J. L., and French, F. S. (1998). Androgen receptorexpression in androgen-independent prostate cancer is associated withincreased expression of androgen-regulated genes. Cancer Res. 58:5718-5724.

[0352] Jarrard, D. F., Sarkar, S., Shi, T., Teager, T. R., Magrane, G.,Kinoshita, H., Nassif, N., Meisner, L., Newton, M. A., and Waldman, F.M. (1999). p16/pRb pathway alterations are required for bypassingsenescence in human prostate epithelial cells. Cancer Res. 59:2957-2964.

[0353] Jenster G. (1999). The role of the androgen receptor in thedevelopment and progression of prostate cancer. Semin. Oncol. 26:407-421.

[0354] Koivisto, P., Kolmer, M., Visakorpi, T., and Kallioniemi O. P.(1996). Androgen receptor gene and hormonal therapy failure of prostatecancer. Am. J. Pathol. 152: 1-9.

[0355] Korn, W. M., Yasutake, T., Kuo, W. L., Warren, R. S., Collins,C., Tomita, M., Gray, J., and Waldman, F. M. (1999). Chromosome arm 20qgains and other genomic alterations in colorectal cancer metastatic toliver, as analyzed by comparative genomic hybridization and fluorescencein situ hybridization. Genes Chromosomes Cancer. 25: 82-90.

[0356] Lin, B., Ferguson, C., White, J. T., Wang, S., Vessella, R.,True, L. D., Hood, L., and Nelson, P. (1999). Prostate-localized andandrogen-regulated expression of the membrane-bound serine proteaseTMPRSS2. Cancer Res. 59: 4180-4184.

[0357] Mahlamaki, E. H., Hoglund, M., Gorunova, L., Karhu, R.,Dawiskiba, S., AndrenSandberg, A., Kallioniemi, P. P., and Johansson, B.(1997). Comparative genomic hybridization reveals frequent gains of 20q,8q, 11q, 12p, and 17q, and losses of 18q, 9p, and 15q in pancreaticcancer. Genes Chromosomes Cancer. 24: 383-391.

[0358] Moul J. W. (1998). Contemporary hormonal management of advancedprostate cancer. Oncology, 12: 499-505.

[0359] Nagabhushan, M., Miller, C. M., Pretlow, T. P., Ciacomia, J. M.,Edgehouse, N. L., Schwarts, S., Kung, H., White, R. W., Gumerlock, P.H., Resnick, M. I., Amini, S. B., and Pretlow, T. G. (1996). CWR22: thefirst human prostate cancer xenograft with strongly androgen-dependentand relapsed strains both in vivo and in soft agar. Cancer Res. 56:3042-3046.

[0360] Richter, J., Beffa, L., Wagner, U., Schraml, P., Gasser, T. C.,Moch, H., Mihatsch, M. J., and Sauter, G. (1998). Patterns ofchromosomal imbalances in advanced urinary bladder cancer detected bycomparative genomic hybridization. Am. J. Pathol. 153: 1615-1621.

[0361] Stubbs, A. P., Abel, P. D., Golding, M., Bhangal, G., Wang, Q.,Waxman, J., Stamp, G. W., and Lalani, E. N. (1999). Differentiallyexpressed genes in hormone refractory prostate cancer: association withchromosomal regions involved with genetic aberrations. Am. J. Pathol.154: 1335-1343.

[0362] Tanner, M. M., Tirkkonen, M., Kallioniemi, A., Isola, J.,Kuukasjarvi, T., Collins, C., Kowbel, D., Guan, X. Y., Trent, J., andGray, J. W. (1996). Independent amplification and frequentco-amplification of three nonsyntenic regions on the long arm ofchromosome 20 in human breast cancer. Cancer Res. 56: 3441-3445.

[0363] Zhang, L., Zhou, W., Velculescu, V. E., Kern, S. E., Hruban, R.H., Hamilton, S. R., Vogelstein, B., And Kinzler, K. W. (1997). Geneexpression profiles in normal and cancer cells. Science, 276: 1268-1272.

[0364] Douarin, B. L., You, J., Nielsen, A. L., Chambon, P., and Losson,R., Tif1α: a possible link between KRAB zinc finger proteins and nuclearreceptors. J. Steroid Biochem. Molec. Biol., 65, 43-50 (1998).

[0365] Xu, L., Su, Y., Labiche, R., Mcleod, D. G., Moul, J. W., andSrivastava, S., Quantitative Evaluation of the Expression Profile of theAndrogen Regulated Genes (ARGs) in Prostate Cancer Cells. AACR annualmeeting (1999).

[0366] Xu, L., Glass, C. K., and Rosenfeld, M. G., Coactivator andcorepressor complexes in nuclear receptor function. Curr. Opin. Genet.Dev., 9, 140-147 (1999).

[0367] Miyajima, N., Kadowaki, Y., Fukushige, S., Shimizu, S., Semba,K., Yamanashi, Y., Matsubara, K., Toyoshima, K., and Yamamoto, T.,Identification of two novel members of erbA superfamily by molecularcloning: the gene products of the two are highly related to each other.Nucleic Acids Res., 16, 11057-11074 (1998).

[0368] Sreenath, T., Orosz, A., Fujita, K., and Bieberich, C. J.,Androgen-independent expression of hoxb-13 in the mouse prostate.Prostate, 41, 203-207 (1999).

[0369] Patel, M. S., and Harris, R. A., Mammalian alpha-keto aciddehydrogenase complexes: gene regulation and genetic defects. FASEB J.,9, 1164-1172 (1995).

[0370] Ho, L., Wexler, I. D., Liu, T. C., Thekkumkara, T. J., and Patel,M. S., Characterization of cDNAs encoding human pyruvate dehydrogenasealpha subunit. Proc. Nat. Acad. Sci., 86, 5330-5334 (1989).

[0371] Ton, C., Hwang, D. M., Dempsey, A. A., and Liew, C. C.,Identification and primary structure of five human NADH-ubiquinoneoxidoreductase subunits. Biochem. Biophys. Res. Commun., 241, 589-594(1997).

[0372] Blachly-Dyson, E., Baldini, A., Litt, M., Mccabe, E. R. B., andForte, M., Human genes encoding the voltage-dependent anion channel(VDAC) of the outer mitochondrial membrane: mapping and identificationof two new isoforms. Genomics, 20, 62-67 (1994).

[0373] Swinnen, J. V., Vercaeren, I., Esquenet, M., Heyns, W., andVerhoeven, G., Androgen regulation of the messenger RNA encodingdiazepam-binding inhibitor/acyl-CoA-binding protein in the rat. Mol.Cell Endocrinol., 118, 65-70 (1996).

[0374] Knudsen, J., Mandrup, S., Rasmussen, J. T., Andreasen, P. H.,Poulsen, F., and Kristiansen, K., The function of acyl-CoA-bindingprotein (ACBP)/diazepam binding inhibitor (DBI). Mol. Cell Biochem.,123, 129-138 (1993).

[0375] Miranda-Vizuete, A., Gustafsson, J. A., and Spyrou, G., Molecularcloning and expression of a cDNA encoding a human thioredoxin-likeprotein. Biochem. Biophys. Res. Commun., 243, 284-288 (1998).

[0376] Cartwright, R., Tambini, C. E., Simpson, P. J., and Thacker, J.,The XRCC2 DNA repair gene from human and mouse encodes a novel member ofthe recA/RAD51 family. Nucleic Acids Res., 26, 3084-3089 (1998).

[0377] Umbricht, C. B., Erdile, L. F., Jabs, E. W., and Kelly, T. J.,Cloning, overexpression, and genomic mapping of the 14-kDa subunit ofhuman replication protein A. J. Biol. Chem., 268, 6131-6138 (1993).

[0378] Gu, Z., Flemington, C., Chittenden, T., and Zambetti, G. P.,ei24, a p53 response gene involved in growth suppression and apoptosis.Mol. Cell. Biol., 20, 233-241 (2000).

[0379] Srivastava, M., and Pollard, H. B., Molecular dissection ofnucleolin's role in growth and cell proliferation: new insights. FASEBJ., 13, 1911-1922 (1999).

[0380] Page-Mccaw, P. S., Amonlirdviman, K., and Sharp, P. A., Puf60: AU2AF65 homolog that binds the pyrimidine tract. RNA, 5, 1548-1560(1999).

[0381] Qian, Z., and Wilusz, J., Grsf-1: a poly (A)+ mRNA bindingprotein which interacts with a conserved G-rich element. Nucleic AcidsRes., 22, 2334-2343 (1994).

[0382] Craig, A. W., Haghighat, A., Yu, A. T., and Sonenberg, N.,Interaction of polyadenylate-binding protein with the eIF4G homologuePAIP enhances translation. Nature, 392, 520-523 (1998).

[0383] Hunt, S. L., Hsuan, J. J., Totty, N., and Jackson, R. J., unr, acellular cytoplasmic RNAbinding protein with five cold-shock domains, isrequired for internal initiation of translation of human rhinovirus RNA.Genes Dev., 13, 437-448 (1999).

[0384] Velculescu, V. E., Zhang, L., Zhou, W., Vogelstein, J., Basrai,M. A., Bassett, D. E. Jr., Hieter, P., Vogelstein, B., and Kinzler, K.W., Characterization of the yeast transcriptome. Cell, 88, 243-251(1997).

[0385] Polyak, K., Xia, Y., Zweier, J. L., Kinzler, K. W., andVogelstein, B., A model for p53-induced apoptosis. Nature, 389, 300-305(1997).

[0386] Hermeking, H., Lengauer, C., Polyak, K., He, T. C., Zhang, L.,Thiagalingam, S., Kinzler, K. W., and Vogelstein, B. 14-3-3-σ is ap53-regulated inhibitor of G2/M progression. Molecular Cell, 1, 3-11(1997).

[0387] Korinek, V., Barker, N., Morin, P. J., Wichen, D., Weger, R.,Kinzler, K. W., Vogelstein, B., and Clevers, H., Constitutivetranscriptional activation by a β-Catenin-Tcf complex in APC^(−/−) coloncarcinoma. Science, 275, 1784-1787 (1997).

[0388] Zhang, L., Zhou, W., Velculescu, V. E., Kern, S. E., Hruban, R.H., Hamilton, S. R., Vogelstein, B., and Kinzler, K. W., Gene expressionprofiles in normal and cancer cells. Science, 276, 1268-1272 (1997).

[0389] Hibi, K., Liu, Q., Beaudry, G. A., Madden, S I., Westra, W. H.,Wehage, S. L., Yang, S. C., Heitmiller, R. F., Bertelsen, A. H.,Sidransky, D., and Jen, J. Serial analysis of gene expression innon-small cell lung cancer. Cancer Res., 58, 5690-5694 (1998).

[0390] Nacht, M., Ferguson, A. T., Zhang, W., Petroziello, J. M., Cook,B. P., Gao, Y. H., Maguire, S., Riley, D., Coppola, G., Landes, G. M.,Madden, S. L., and Sukumar, S., Combining serial analysis of geneexpression and array technologies to identify genes differentiallyexpressed in breast cancer. Cancer Res., 59, 5464-5470 (1999).

[0391] Waard, V., Berg, B. M. M., Veken, J., Schultz-Heienbrok, R.,Pannekoek, H., and Zonneveld, A., Serial analysis of gene expression toasssess the endothelial cell response to an atherogenic stimulus. Gene,226, 1-8 (1999).

[0392] Berg, A., Visser, L., and Poppema, S., High expression of the CCchemokine TARC in reed-sternberg cells. A possible explanation for thecharacteristic T-cell infiltrate in hodgkin” lymphoma. Am. J. Pathol.,154, 1685-1691 (1999).

[0393] Iyer, V. R., Eisen, M. B., Ross, D. T., Schuler, G., Moore, T.,Lee, J. C. F., Trent, J. M., Staudt, L. M., Hudson, J. Jr., Boguski, M.S., Lashkari, D., Shalon, D., Botstein, D., and Brown, P. O., Thetrancriptional program in the response of human fibroblasts to serum.Science, 283, 83-87 (1999).

[0394] Charpentier, A. H., Bednarek, A. K., Daniel, R. L., Hawkins, K.A., Laflin, K. J., Gaddis, S., Macleod, M. C., and Aldaz, C. M., Effectsof estrogen on global gene expression: identification of novel targetsof estrogen action. Cancer Res., 60, 5977-5983 (2000).

[0395] Ripple, M. O., Henry, W. F., Rago, R. P., and Wilding, G.,Prooxidant-antioxidant shift induced by androgen treatment of humanprostate carcinoma cells. J. Nat. Cancer Inst., 89, 40-48 (1997).

1 81 1 1140 DNA Homo sapiens CDS (95)..(850) 1 tccttgggtt cgggtgaaagcgcctggggg ttcgtggcca tgatccccga gctgctggag 60 aactgaaggc ggacagtctcctgcgaaaca ggca atg gcg gag ctg gag ttt gtt 115 Met Ala Glu Leu Glu PheVal 1 5 cag atc atc atc atc gtg gtg gtg atg atg gtg atg gtg gtg gtg atc163 Gln Ile Ile Ile Ile Val Val Val Met Met Val Met Val Val Val Ile 1015 20 acg tgc ctg ctg agc cac tac aag ctg tct gca cgg tcc ttc atc agc211 Thr Cys Leu Leu Ser His Tyr Lys Leu Ser Ala Arg Ser Phe Ile Ser 2530 35 cgg cac agc cag ggg cgg agg aga gaa gat gcc ctg tcc tca gaa gga259 Arg His Ser Gln Gly Arg Arg Arg Glu Asp Ala Leu Ser Ser Glu Gly 4045 50 55 tgc ctg tgg ccc tcg gag agc aca gtg tca ggc aac gga atc cca gag307 Cys Leu Trp Pro Ser Glu Ser Thr Val Ser Gly Asn Gly Ile Pro Glu 6065 70 ccg cag gtc tac gcc ccg cct cgg ccc acc gac cgc ctg gcc gtg ccg355 Pro Gln Val Tyr Ala Pro Pro Arg Pro Thr Asp Arg Leu Ala Val Pro 7580 85 ccc ttc gcc cag cgg gag cgc ttc cac cgc ttc cag ccc acc tat ccg403 Pro Phe Ala Gln Arg Glu Arg Phe His Arg Phe Gln Pro Thr Tyr Pro 9095 100 tac ctg cag cac gag atc gac ctg cca ccc acc atc tcg ctg tca gac451 Tyr Leu Gln His Glu Ile Asp Leu Pro Pro Thr Ile Ser Leu Ser Asp 105110 115 ggg gag gag ccc cca ccc tac cag ggc ccc tgc acc ctc cag ctt cgg499 Gly Glu Glu Pro Pro Pro Tyr Gln Gly Pro Cys Thr Leu Gln Leu Arg 120125 130 135 gac ccc gag cag cag ctg gaa ctg aac cgg gag tcg gtg cgc gcaccc 547 Asp Pro Glu Gln Gln Leu Glu Leu Asn Arg Glu Ser Val Arg Ala Pro140 145 150 cca aac aga acc atc ttc gac agt gac ctg atg gat agt gcc aggctg 595 Pro Asn Arg Thr Ile Phe Asp Ser Asp Leu Met Asp Ser Ala Arg Leu155 160 165 ggc ggc ccc tgc ccc ccc agc agt aac tcg ggc atc agc gcc acgtgc 643 Gly Gly Pro Cys Pro Pro Ser Ser Asn Ser Gly Ile Ser Ala Thr Cys170 175 180 tac ggc agc ggc ggg cgc atg gag ggg ccg ccg ccc acc tac agcgag 691 Tyr Gly Ser Gly Gly Arg Met Glu Gly Pro Pro Pro Thr Tyr Ser Glu185 190 195 gtc atc ggc cac tac ccg ggg tcc tcc ttc cag cac cag cag agcagt 739 Val Ile Gly His Tyr Pro Gly Ser Ser Phe Gln His Gln Gln Ser Ser200 205 210 215 ggg ccg ccc tcc ttg ctg gag ggg acc cgg ctc cac cac acacac atc 787 Gly Pro Pro Ser Leu Leu Glu Gly Thr Arg Leu His His Thr HisIle 220 225 230 gcg ccc cta gag agc gca gcc atc tgg agc aaa gag aag gataaa cag 835 Ala Pro Leu Glu Ser Ala Ala Ile Trp Ser Lys Glu Lys Asp LysGln 235 240 245 aaa gga cac cct ctc tagggtcccc aggggggccg ggctggggctgcgtaggtga 890 Lys Gly His Pro Leu 250 aaaggcagaa cactccgcgc ttcttagaagaggagtgaga ggaaggcggg gggcgcagca 950 acgcatcgtg tggccctccc ctcccacctccctgtgtata aatatttaca tgtgatgtct 1010 ggtctgaatg cacaagctaa gagagcttgcaaaaaaaaaa agaaaaaaga aaaaaaaaaa 1070 ccacgtttct ttgttgagct gtgtcttgaaggcaaaagaa aaaaaatttc tacagtaaaa 1130 aaaaaaaaaa 1140 2 759 DNA Homosapiens 2 atggcggagc tggagtttgt tcagatcatc atcatcgtgg tggtgatgatggtgatggtg 60 gtggtgatca cgtgcctgct gagccactac aagctgtctg cacggtccttcatcagccgg 120 cacagccagg ggcggaggag agaagatgcc ctgtcctcag aaggatgcctgtggccctcg 180 gagagcacag tgtcaggcaa cggaatccca gagccgcagg tctacgccccgcctcggccc 240 accgaccgcc tggccgtgcc gcccttcgcc cagcgggagc gcttccaccgcttccagccc 300 acctatccgt acctgcagca cgagatcgac ctgccaccca ccatctcgctgtcagacggg 360 gaggagcccc caccctacca gggcccctgc accctccagc ttcgggaccccgagcagcag 420 ctggaactga accgggagtc ggtgcgcgca cccccaaaca gaaccatcttcgacagtgac 480 ctgatggata gtgccaggct gggcggcccc tgccccccca gcagtaactcgggcatcagc 540 gccacgtgct acggcagcgg cgggcgcatg gaggggccgc cgcccacctacagcgaggtc 600 atcggccact acccggggtc ctccttccag caccagcaga gcagtgggccgccctccttg 660 ctggagggga cccggctcca ccacacacac atcgcgcccc tagagagcgcagccatctgg 720 agcaaagaga aggataaaca gaaaggacac cctctctag 759 3 252 PRTHomo sapiens 3 Met Ala Glu Leu Glu Phe Val Gln Ile Ile Ile Ile Val ValVal Met 1 5 10 15 Met Val Met Val Val Val Ile Thr Cys Leu Leu Ser HisTyr Lys Leu 20 25 30 Ser Ala Arg Ser Phe Ile Ser Arg His Ser Gln Gly ArgArg Arg Glu 35 40 45 Asp Ala Leu Ser Ser Glu Gly Cys Leu Trp Pro Ser GluSer Thr Val 50 55 60 Ser Gly Asn Gly Ile Pro Glu Pro Gln Val Tyr Ala ProPro Arg Pro 65 70 75 80 Thr Asp Arg Leu Ala Val Pro Pro Phe Ala Gln ArgGlu Arg Phe His 85 90 95 Arg Phe Gln Pro Thr Tyr Pro Tyr Leu Gln His GluIle Asp Leu Pro 100 105 110 Pro Thr Ile Ser Leu Ser Asp Gly Glu Glu ProPro Pro Tyr Gln Gly 115 120 125 Pro Cys Thr Leu Gln Leu Arg Asp Pro GluGln Gln Leu Glu Leu Asn 130 135 140 Arg Glu Ser Val Arg Ala Pro Pro AsnArg Thr Ile Phe Asp Ser Asp 145 150 155 160 Leu Met Asp Ser Ala Arg LeuGly Gly Pro Cys Pro Pro Ser Ser Asn 165 170 175 Ser Gly Ile Ser Ala ThrCys Tyr Gly Ser Gly Gly Arg Met Glu Gly 180 185 190 Pro Pro Pro Thr TyrSer Glu Val Ile Gly His Tyr Pro Gly Ser Ser 195 200 205 Phe Gln His GlnGln Ser Ser Gly Pro Pro Ser Leu Leu Glu Gly Thr 210 215 220 Arg Leu HisHis Thr His Ile Ala Pro Leu Glu Ser Ala Ala Ile Trp 225 230 235 240 SerLys Glu Lys Asp Lys Gln Lys Gly His Pro Leu 245 250 4 8 PRT ArtificialSequence Description of Artificial Sequence FLAG peptide 4 Asp Tyr LysAsp Asp Asp Asp Lys 1 5 5 24 DNA Artificial Sequence Description ofArtificial Sequence Primer 5 ggcagaacac tccgcgcttc ttag 24 6 24 DNAArtificial Sequence Description of Artificial Sequence Primer 6caagctctct tagcttgtgc attc 24 7 22 DNA Artificial Sequence Descriptionof Artificial Sequence Primer 7 cttgggttcg ggtgaaagcg cc 22 8 22 DNAArtificial Sequence Description of Artificial Sequence Primer 8ggtgggtggc aggtcgatct cg 22 9 20 DNA Artificial Sequence Description ofArtificial Sequence Primer 9 ccttcgccca gcgggagcgc 20 10 24 DNAArtificial Sequence Description of Artificial Sequence Primer 10caagctctct tagcttgtgc attc 24 11 249 PRT Homo sapiens 11 Ala Glu Leu GluPhe Val Gln Ile Ile Ile Ile Val Val Val Met Met 1 5 10 15 Val Met ValVal Val Ile Thr Cys Leu Leu Ser His Tyr Lys Leu Ser 20 25 30 Ala Arg SerPhe Ile Ser Arg His Ser Gln Gly Arg Arg Arg Glu Asp 35 40 45 Ala Leu SerSer Glu Gly Cys Leu Trp Pro Ser Glu Ser Thr Val Ser 50 55 60 Gly Asn GlyIle Pro Glu Pro Gln Val Tyr Ala Pro Pro Arg Pro Thr 65 70 75 80 Asp ArgLeu Ala Val Pro Pro Phe Ala Gln Arg Glu Arg Phe His Arg 85 90 95 Phe GlnPro Thr Tyr Pro Tyr Leu Gln His Glu Ile Asp Leu Pro Pro 100 105 110 ThrIle Ser Leu Ser Asp Gly Glu Glu Pro Pro Pro Tyr Gln Gly Pro 115 120 125Cys Thr Leu Gln Leu Arg Asp Pro Glu Gln Gln Leu Glu Leu Asn Arg 130 135140 Glu Ser Val Arg Ala Pro Pro Asn Arg Thr Ile Phe Asp Ser Asp Leu 145150 155 160 Met Asp Ser Ala Arg Leu Gly Gly Pro Cys Pro Pro Ser Ser AsnSer 165 170 175 Gly Ile Ser Ala Thr Cys Tyr Gly Ser Gly Gly Arg Met GluGly Pro 180 185 190 Pro Pro Thr Tyr Ser Glu Val Ile Gly His Tyr Pro GlySer Ser Phe 195 200 205 Gln His Gln Gln Ser Ser Gly Pro Pro Ser Leu LeuGlu Gly Thr Arg 210 215 220 Leu His His Thr His Ile Ala Pro Leu Glu SerAla Ala Ile Trp Ser 225 230 235 240 Lys Glu Lys Asp Lys Gln Lys Gly His245 12 244 PRT Homo sapiens 12 Ala Glu Leu Glu Phe Ala Gln Ile Ile IleIle Val Val Val Val Thr 1 5 10 15 Val Met Val Val Val Ile Val Cys LeuLeu Asn His Tyr Lys Val Ser 20 25 30 Thr Arg Ser Phe Ile Asn Arg Pro AsnGln Ser Arg Arg Arg Glu Asp 35 40 45 Gly Leu Pro Gln Glu Gly Cys Leu TrpPro Ser Asp Ser Ala Ala Pro 50 55 60 Arg Leu Gly Ala Ser Glu Ile Met HisAla Pro Arg Ser Arg Asp Arg 65 70 75 80 Phe Thr Ala Pro Ser Phe Ile GlnArg Asp Arg Phe Ser Arg Phe Gln 85 90 95 Pro Thr Tyr Pro Tyr Val Gln HisGlu Ile Asp Leu Pro Pro Thr Ile 100 105 110 Ser Leu Ser Asp Gly Glu GluPro Pro Pro Tyr Gln Gly Pro Cys Thr 115 120 125 Leu Gln Leu Arg Asp ProGlu Gln Gln Met Glu Leu Asn Arg Glu Ser 130 135 140 Val Arg Ala Pro ProAsn Arg Thr Ile Phe Asp Ser Asp Leu Ile Asp 145 150 155 160 Ile Ala MetTyr Ser Gly Gly Pro Cys Pro Pro Ser Ser Asn Ser Gly 165 170 175 Ile SerAla Ser Thr Cys Ser Ser Asn Gly Arg Met Glu Gly Pro Pro 180 185 190 ProThr Tyr Ser Glu Val Met Gly His His Pro Gly Ala Ser Phe Leu 195 200 205His His Gln Arg Ser Asn Ala His Arg Gly Ser Arg Leu Gln Phe Gln 210 215220 Gln Asn Asn Ala Glu Ser Thr Ile Val Pro Ile Lys Gly Lys Asp Arg 225230 235 240 Lys Pro Gly Asn 13 10 DNA Artificial Sequence Description ofArtificial Sequence Synthetic oligonucleotide 13 gccagcccag 10 14 10 DNAArtificial Sequence Description of Artificial Sequence Syntheticoligonucleotide 14 gtgcagggag 10 15 10 DNA Artificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 15gacaaacatt 10 16 10 DNA Artificial Sequence Description of ArtificialSequence Synthetic oligonucleotide 16 atgactcaag 10 17 10 DNA ArtificialSequence Description of Artificial Sequence Synthetic oligonucleotide 17gaaaagaagg 10 18 10 DNA Artificial Sequence Description of ArtificialSequence Synthetic oligonucleotide 18 cctgtacccc 10 19 10 DNA ArtificialSequence Description of Artificial Sequence Synthetic oligonucleotide 19cctgaactgg 10 20 10 DNA Artificial Sequence Description of ArtificialSequence Synthetic oligonucleotide 20 tgacagccca 10 21 10 DNA ArtificialSequence Description of Artificial Sequence Synthetic oligonucleotide 21tacaaaacca 10 22 10 DNA Artificial Sequence Description of ArtificialSequence Synthetic oligonucleotide 22 aattctccta 10 23 10 DNA ArtificialSequence Description of Artificial Sequence Synthetic oligonucleotide 23tgcatatcat 10 24 10 DNA Artificial Sequence Description of ArtificialSequence Synthetic oligonucleotide 24 cttgacacac 10 25 10 DNA ArtificialSequence Description of Artificial Sequence Synthetic oligonucleotide 25tgtctaacta 10 26 10 DNA Artificial Sequence Description of ArtificialSequence Synthetic oligonucleotide 26 gtggacccca 10 27 10 DNA ArtificialSequence Description of Artificial Sequence Synthetic oligonucleotide 27ataaagtaac 10 28 10 DNA Artificial Sequence Description of ArtificialSequence Synthetic oligonucleotide 28 tacattttca 10 29 10 DNA ArtificialSequence Description of Artificial Sequence Synthetic oligonucleotide 29tcagaacagt 10 30 10 DNA Artificial Sequence Description of ArtificialSequence Synthetic oligonucleotide 30 caacttcaac 10 31 10 DNA ArtificialSequence Description of Artificial Sequence Synthetic oligonucleotide 31gataggtcgg 10 32 10 DNA Artificial Sequence Description of ArtificialSequence Synthetic oligonucleotide 32 ctaaaaggag 10 33 10 DNA ArtificialSequence Description of Artificial Sequence Synthetic oligonucleotide 33gtggtgcgtg 10 34 10 DNA Artificial Sequence Description of ArtificialSequence Synthetic oligonucleotide 34 tccccgtggc 10 35 10 DNA ArtificialSequence Description of Artificial Sequence Synthetic oligonucleotide 35attgatcttg 10 36 10 DNA Artificial Sequence Description of ArtificialSequence Synthetic oligonucleotide 36 agctggtttc 10 37 10 DNA ArtificialSequence Description of Artificial Sequence Synthetic oligonucleotide 37cctcccccgt 10 38 10 DNA Artificial Sequence Description of ArtificialSequence Synthetic oligonucleotide 38 atgtactctg 10 39 10 DNA ArtificialSequence Description of Artificial Sequence Synthetic oligonucleotide 39gatgaaatac 10 40 10 DNA Artificial Sequence Description of ArtificialSequence Synthetic oligonucleotide 40 gtgcatcccg 10 41 10 DNA ArtificialSequence Description of Artificial Sequence Synthetic oligonucleotide 41gaaattaggg 10 42 10 DNA Artificial Sequence Description of ArtificialSequence Synthetic oligonucleotide 42 tttctagggg 10 43 10 DNA ArtificialSequence Description of Artificial Sequence Synthetic oligonucleotide 43cccagggaga 10 44 10 DNA Artificial Sequence Description of ArtificialSequence Synthetic oligonucleotide 44 gtggcgcaca 10 45 10 DNA ArtificialSequence Description of Artificial Sequence Synthetic oligonucleotide 45ttgcttttgt 10 46 10 DNA Artificial Sequence Description of ArtificialSequence Synthetic oligonucleotide 46 atgtcctttc 10 47 10 DNA ArtificialSequence Description of Artificial Sequence Synthetic oligonucleotide 47tgtttatcct 10 48 10 DNA Artificial Sequence Description of ArtificialSequence Synthetic oligonucleotide 48 gctttgtatc 10 49 10 DNA ArtificialSequence Description of Artificial Sequence Synthetic oligonucleotide 49gttccagtga 10 50 10 DNA Artificial Sequence Description of ArtificialSequence Synthetic oligonucleotide 50 tagcagaggc 10 51 10 DNA ArtificialSequence Description of Artificial Sequence Synthetic oligonucleotide 51acaaattatg 10 52 10 DNA Artificial Sequence Description of ArtificialSequence Synthetic oligonucleotide 52 cagtttgtac 10 53 10 DNA ArtificialSequence Description of Artificial Sequence Synthetic oligonucleotide 53gattacttgc 10 54 10 DNA Artificial Sequence Description of ArtificialSequence Synthetic oligonucleotide 54 ggccagccct 10 55 10 DNA ArtificialSequence Description of Artificial Sequence Synthetic oligonucleotide 55caattgtaaa 10 56 10 DNA Artificial Sequence Description of ArtificialSequence Synthetic oligonucleotide 56 aaagccaaga 10 57 10 DNA ArtificialSequence Description of Artificial Sequence Synthetic oligonucleotide 57caactaattc 10 58 10 DNA Artificial Sequence Description of ArtificialSequence Synthetic oligonucleotide 58 aagagctaat 10 59 10 DNA ArtificialSequence Description of Artificial Sequence Synthetic oligonucleotide 59cttttcaaga 10 60 10 DNA Artificial Sequence Description of ArtificialSequence Synthetic oligonucleotide 60 gtgtgtaaaa 10 61 10 DNA ArtificialSequence Description of Artificial Sequence Synthetic oligonucleotide 61acaaaatgta 10 62 10 DNA Artificial Sequence Description of ArtificialSequence Synthetic oligonucleotide 62 aaggtagcag 10 63 10 DNA ArtificialSequence Description of Artificial Sequence Synthetic oligonucleotide 63ggcggggcca 10 64 10 DNA Artificial Sequence Description of ArtificialSequence Synthetic oligonucleotide 64 ggccagtaac 10 65 10 DNA ArtificialSequence Description of Artificial Sequence Synthetic oligonucleotide 65aacttaagag 10 66 10 DNA Artificial Sequence Description of ArtificialSequence Synthetic oligonucleotide 66 agggatggcc 10 67 10 DNA ArtificialSequence Description of Artificial Sequence Synthetic oligonucleotide 67cttaaggatt 10 68 243 PRT Mus sp. 68 Ile Thr Glu Leu Glu Phe Val Gln IleVal Val Ile Val Val Val Met 1 5 10 15 Met Val Met Val Val Met Ile ThrCys Leu Leu Ser His Tyr Lys Leu 20 25 30 Ser Ala Arg Ser Phe Ile Ser ArgHis Ser Gln Ala Arg Arg Arg Asp 35 40 45 Asp Gly Leu Ser Ser Glu Gly CysLeu Trp Pro Ser Glu Ser Thr Val 50 55 60 Ser Gly Gly Met Pro Glu Pro GlnVal Tyr Ala Pro Pro Arg Pro Thr 65 70 75 80 Asp Arg Leu Ala Val Pro ProPhe Ile Gln Arg Ser Arg Phe Gln Pro 85 90 95 Thr Tyr Pro Tyr Leu Gln HisGlu Ile Ala Leu Pro Pro Thr Ile Ser 100 105 110 Leu Ser Asp Gly Glu GluPro Pro Pro Tyr Gln Gly Pro Cys Thr Leu 115 120 125 Gln Leu Arg Asp ProGlu Gln Gln Leu Glu Leu Asn Arg Glu Ser Val 130 135 140 Arg Ala Pro ProAsn Arg Thr Ile Phe Asp Ser Asp Leu Ile Asp Ser 145 150 155 160 Thr MetLeu Gly Gly Pro Cys Pro Pro Ser Ser Asn Ser Gly Ile Ser 165 170 175 AlaThr Cys Tyr Ser Ser Gly Gly Arg Met Glu Gly Pro Pro Pro Thr 180 185 190Tyr Ser Glu Val Ile Gly His Tyr Pro Gly Ser Ser Phe Gln His Gln 195 200205 Gln Ser Asn Gly Pro Ser Ser Leu Leu Glu Gly Thr Arg Leu His His 210215 220 Ser His Ile Ala Pro Leu Glu Asn Lys Glu Lys Glu Lys Gln Lys Gly225 230 235 240 His Pro Leu 69 21 DNA Artificial Sequence Description ofArtificial Sequence Primer 69 gctgctggag aactgaaggc g 21 70 22 DNAArtificial Sequence Description of Artificial Sequence Primer 70gtgtcctttc tgtttatcct tc 22 71 27 DNA Artificial Sequence Description ofArtificial Sequence Primer 71 aagcttgctg ctggagaact gaaggcg 27 72 25 DNAArtificial Sequence Description of Artificial Sequence Primer 72gaattcggtg tcctttctgt ttatc 25 73 27 DNA Artificial Sequence Descriptionof Artificial Sequence Primer 73 gcaggatccc aaccagatgc tgcttgc 27 74 28DNA Artificial Sequence Description of Artificial Sequence Primer 74gcagaattct tttgtaatcc ctggagta 28 75 27 DNA Artificial SequenceDescription of Artificial Sequence Primer 75 gcaaagcttg tccggtttgctggaagc 27 76 31 DNA Artificial Sequence Description of ArtificialSequence Primer 76 gcagaattcc ctttttgttc ttattggtga c 31 77 18 DNAArtificial Sequence Description of Artificial Sequence Primer 77catgatcccc gagctgct 18 78 23 DNA Artificial Sequence Description ofArtificial Sequence Primer 78 tgatctgaac aaactccagc tcc 23 79 23 DNAArtificial Sequence Description of Artificial Sequence Primer 79aggcggacag tctcctgcga aac 23 80 4 PRT Artificial Sequence Description ofArtificial Sequence Synthetic motif 80 Pro Pro Pro Tyr 1 81 4 PRTArtificial Sequence Description of Artificial Sequence Synthetic motif81 Pro Pro Thr Tyr 1

We claim:
 1. A polypeptide, wherein the polypeptide comprises an aminoacid sequence that is at least 95% identical to SEQ ID NO:3 and whereinthe polypepide inhibits the growth of LNCaP cells in a colony-formingassay.
 2. A polypeptide variant of SEQ ID NO:3, wherein the variantcomprises at least one mutation and/or deletion in at least one of thePY motifs of SEQ ID NO:3.
 3. An isolated nucleic acid, wherein thenucleic acid hybridizes to a DNA having the nucleotide sequence of SEQID NO:2 under conditions of high stringency, wherein the nucleic acidencodes a polypeptide that inhibits the growth of LNCaP cells in acolony-forming assay.
 4. An isolated antibody that binds to thepolypeptide of claim
 1. 5. An isolated antibody that binds to thepolypeptide of claim
 2. 6. An isolated antibody that binds to thepolypeptide of claim
 3. 7. A method of reducing the expression of anandrogen receptor in a prostate cancer cell comprising administering apolypeptide according to claim 1 to the prostate cancer cell in anamount effective to reduce expression of the androgen receptor in thecell.
 8. A method of inhibiting the growth of a prostate cancer cell,comprising administering a polypeptide according to claim 1 to theprostate cancer cell in an amount effective to inhibit the growth of thecancer cell.
 9. A method of modulating the expression of a gene in aprostate cancer cell, wherein transcription of the gene is regulated byan androgen receptor, comprising administering a polypeptide accordingto claim 1 to the prostate cancer cell in an amount effective tomodulate the expression of the gene in the cell.